Nanomedicines for early nerve repair

ABSTRACT

Disclosed are hydrophobically modified nanoparticles and polymeric nanostructures that can be utilized to for the treatment of neuronal injury or neuronal disease in an affected patient, and methods of forming and using the nanoparticles and nanostructures.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.14/001,189 filed Aug. 23, 2013, which is a national application ofInternational Application No. PCT/US12/26590 filed Feb. 24, 2012, whichclaims the benefit of: U.S. Provisional Application 61/446,252 filedFeb. 24, 2011; U.S. Provisional Application 61/879,248 filed Sep. 18,2013; and, U.S. Provisional Application 61/879,249 filed Sep. 18, 2013each of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

This invention generally pertains to the field of nanomedicine. Moreparticularly, the invention pertains to hydrophobically modifiednanoparticles and methods of forming and using the same.

BACKGROUND OF THE INVENTION

Neural injuries and neural diseases are debilitating and complexmanifestations of the body. For example, spinal cord injury (SCI)results in immediate initial disruption of cell membranes in affectedneural and endothelial tissues, followed by extensive secondaryneurodegenerative processes. Most SCI cases involve a primary injury anda subsequent secondary damage. During the primary injury, the acutemechanical stress to the spinal cord breaks neural membranes and causesCa²⁺ influx into cells. The latter processes trigger a series ofsecondary biological events including inflammation, free radicalrelease, and apoptosis, which further exacerbate the damage.

Among various treatments under investigation, a key approach is to sealthe damaged membrane at the early stage of SCI. To date, poly(ethyleneglycol) (PEG) and Pluronic P188 have been used for membrane repair.However, the effectiveness of these agents has been very limited partlydue to their rapid clearance after systemic administration. For example,a PEG without hydrophobic modification can result in negligible efficacyof the treatment of neural injuries.

There is a need in the art for new compositions and methods for treatingneural injuries. The present invention addresses that need.

BRIEF SUMMARY OF THE INVENTION

Applicant has demonstrated a function of block copolymer micelles as ananoscale membrane repair agent in traumatically injured spinal cord.Axonal membranes injured by compression may be effectively repaired byself-assembled monomethoxy poly(ethylene glycol)-poly(D,L-lactic acid)(mPEG-PDLLA) di-block copolymer micelles (10 nm to 100 nm in diameter).Intravenously injected mPEG-PDLLA micelles recover locomotor functionand reduce the volume and inflammatory response of the lesion in SCIrats. Mechanistically, it is believed that copolymers with controlledamphiphilic properties are able to insert the hydrophobic chain into amechanically disrupted membrane which has a lower density of lipidpacking, but are repelled after the membrane is sealed. However, in vivodecomposition of the self-assembled micelles during systemic circulationpermits effective delivery of amphiphilic unimers to the injury site.

Polymer micelles are designed to encapsulate hydrophobicanti-inflammatory drugs that effectively suppress the intracellularinjury induced by Ca²⁺ influx. After systemic administration, micellesreduce their stability during blood circulation, as shown by FRETstudies, especially when the loaded drug is released. Both unimers andanti-inflammatory drugs are delivered to the injury site through thecompromised blood-spinal cord barrier.

In addition, administration of methylprednisolone, the only clinicallyapproved neuroprotective drug for treating acute SCI, is controversialbecause of the high dosages of methylprednisolone that are required toachieve therapeutic levels at the injury site. High doses ofmethylprednisolone are required due to its low bioavailability at theinjury site, a factor that is related to both poor solubility of drugand the drug's difficulty in crossing the blood spinal cord barrier. Asa result, an extremely high dose of methylprednisolone is required,often leading to systemic toxicity in patients.

Furthermore, the polymer micelle-based membrane repair method ischallenged by the narrow therapeutic time window in clinicalapplications. For instance, the micelles have to be administered beforethe secondary neuronal injury becomes dominant. Thus, an alternativetreatment option for the treatment of neural injuries and diseases ishighly desired.

The present disclosure demonstrates that problems for the treatment ofneural injuries and diseases can be overcome using therapeuticcompositions with dual actions of 1) repair of damaged membrane and 2)suppression of intracellular inflammation action. The describedcompositions may act synergistically to rescue more neural cells frominjury induced cell death and further extend the therapeutic window forintervention as compared to treatments having only a single action.

The present disclosure describes hydrophobically modified nanoparticlesthat can be utilized for the treatment of neural injury or neuraldisease in an affected patient, along with methods of forming and usingthe nanoparticles.

The hydrophobically modified nanoparticles according to the presentdisclosure provide several advantages compared to alternatives known inthe art. First, the nanoparticles of the present disclosure are designedas “dual action” compositions to treat neural injury via repair ofdamaged membrane and suppression of intracellular inflammation.

Second, the nanoparticles of the present disclosure are capable ofproviding both “burst” effects and “extended” effects of thepharmacophore of the described compositions. As a result, a lower doseof the pharmacophore (e.g., a steroid) may be achieved, while stillproviding effective treatment.

Third, the nanoparticles of the present disclosure have improvedpharmacokinetic parameters compared to alternatives known in the art.For example, the nanoparticles may be associated with a more targeteddelivery to the site in need of repair or treatment, and may beassociated with a reduction in potentially harmful side effects and/ortoxicities at other sites of the body.

Fourth, compared to other ethylene glycol embodiments used in the art,the nanoparticles of the present disclosure are hydrophobically modifiedor include a hydrophobic domain, respectively. The inclusion of ahydrophobic moiety enhances the effectiveness of the compositions due toa slower rate of clearance from the body after systemic administration.

Fifth, in the embodiments in which the nanoparticles of the presentdisclosure include an anti-inflammatory agent, the resultant compositionmay be administered to a patient as a single agent without the need forseparate administrations of the nanoparticles and the anti-inflammatoryagent.

Finally, the nanoparticles of the present disclosure may have improvedloading efficiency of an anti-inflammatory agent in order to facilitatea more potent and targeted delivery of the anti-inflammatory agent tothe site in need of repair or treatment.

The following numbered embodiments are contemplated and arenon-limiting:

1. A composition comprising a hydrophobically modified nanoparticlecomprising a polysaccharide and a pharmacophore, wherein thepolysaccharide is covalently bound to the pharmacophore.

2. The composition of clause 1 or clause 2 wherein the polysaccharide iscovalently bound to the pharmacophore via an amide bond.

3. The composition of clause 1 or clause 2 wherein the polysaccharide ischitosan.

4. The composition of clause 1 or clause 2 wherein the polysaccharide isa chitosan derivative.

5. The composition of clause 1 or clause 2 wherein the polysaccharide isglycol chitosan.

6. The composition of any one of clauses 1 to 5 wherein thepharmacophore is a fatty acid.

7. The composition of any one of clauses 1 to 5 wherein thepharmacophore is cholanic acid.

8. The composition of any one of clauses 1 to 5 wherein thepharmacophore is ferulic acid.

9. The composition of any one of clauses 1 to 5 wherein thepharmacophore is a ferulic acid derivative.

10. The composition of clause 1 wherein the polysaccharide is glycolchitosan and the pharmacophore is ferulic acid.

11. The composition of clause 10 wherein the nanoparticle has a degreeof substitution of ferulic acid per glycol chitosan (ferulic acid:glycolchitosan chain) selected from the group consisting of 5:1, 11:1, and21:1.

12. The composition of clause 10 wherein the nanoparticle has a degreeof substitution of ferulic acid per glycol chitosan (ferulic acid:glycolchitosan chain) of 11:1.

13. The composition of any one of clauses 1 to 12 further comprising atherapeutically effective amount of an anti-inflammatory agent.

14. The composition of clause 13 wherein the anti-inflammatory agent isa corticosteroid.

15. The composition of clause 14 wherein the corticosteroid is selectedfrom the group consisting of betamethasone, dexamethasone, flumethasone,methylprednisolone, paramethasone, prednisolone, prednisone,triamcinolone, hydrocortisone, and cortisone.

16. The composition of clause 15 wherein the corticosteroid ismethylprednisolone.

17. The composition of any one of clauses 1 to 16 wherein thepharmacophore is methylprednisolone (MP).

18. The composition of any one of clauses 1 to 16 wherein thepharmacophore is methylprednisolone hemisuccinate (MPHS).

19. The composition of clause 1 wherein the polysaccharide is glycolchitosan and the pharmacophore is methylprednisolone.

20. The composition of clause 1 wherein the polysaccharide is glycolchitosan and the pharmacophore is methylprednisolone hemisuccinate.

21. The composition of clause 20 wherein the nanoparticle has a degreeof substitution of methylprednisolone per glycol chitosan(methylprednisolone:glycol chitosan chain) is in the range of from about5:1 to about 21:1.

22. The composition of clause 20 wherein the nanoparticle has a degreeof substitution of methylprednisolone per glycol chitosan(methylprednisolone:glycol chitosan chain) of 11:1.

23. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanostructure is about 10 nm to about 950 nanometers(nm).

24. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 100 nm to about 950 nm.

25. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 100 nm to about 500 nm.

26. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 100 nm to about 400 nm.

27. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 100 nm to about 200 nm.

28. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 200 nm to about 400 nm.

29. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 250 nm to about 350 nm.

30. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 300 nm to about 400 nm.

31. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 10 nm to about 200 nm.

32. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 10 nm to about 150 nm.

33. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 10 nm to about 100 nm.

34. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 10 nm to about 50 nm.

35. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 100 nanometers.

36. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 150 nanometers.

37. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 200 nanometers.

38. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 250 nanometers.

39. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 300 nanometers.

40. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 320 nanometers.

41. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 350 nanometers.

42. The composition of any one of clauses 1 to 2 wherein the averagediameter of the nanoparticle is about 400 nanometers.

43. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 450 nanometers.

44. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 500 nanometers.

45. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 550 nanometers.

46. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 600 nanometers.

47. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 650 nanometers.

48. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 700 nanometers.

49. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 750 nanometers.

50. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 800 nanometers.

51. The composition of any one of clauses 1 to 22 wherein the averagediameter of the nanoparticle is about 900 nanometers.

52. The composition of any one of clauses 1 to 51 wherein thecomposition is a micelle.

53. A method of treating a patient having a neuronal injury, the methodcomprising the step of administering to the patient a therapeuticallyeffective amount of the composition of any one of clauses 1 to 52.

54. The method of clause 53 wherein the neuronal injury is a spinal cordinjury.

55. The method of clause 53 wherein the neuronal injury is a traumaticbrain injury.

56. The method of clause 53 wherein the neuronal injury is an acuteneuronal injury.

57. The method of clause 53 wherein the neuronal injury is a cranialneuronal injury.

58. The method of any one of clauses 53 to 56 wherein the neuronalinjury results in hearing loss of the patient.

59. The method of any one of clauses 53 to 56 wherein the neuronalinjury results in vertigo of the patient.

60. The method of any one of clauses 53 to 56 wherein the neuronalinjury results in loss of equilibrium of the patient.

61. The method of any one of clauses 53 to 56 wherein the neuronalinjury results in nystagmus of the patient.

62. The method of any one of clauses 53 to 56 wherein the neuronalinjury results in motion sickness of the patient.

63. The method of any one of clauses 53 to 56 wherein the neuronalinjury results in tinnitus of the patient.

64. The method of clause 58 wherein the hearing loss is due tonoise-induced nerve damage.

65. The method of any one of clauses 53 to 63 wherein the neuronalinjury is a damaged tympanic membrane.

66. The method of any one of clauses 53 to 63 wherein the neuronalinjury is a damaged cranial nerve injury.

67. The method of clause 66 wherein the cranial nerve is thevestibulocochlear nerve.

68. The method of clause 67 wherein the vestibulocochlear nervecomprises the cochlear nerve.

69. The method of clause 67 wherein the vestibulocochlear nervecomprises the vestibular nerve.

70. The method of clause 66 wherein the cranial nerve is the olfactorynerve.

71. The method of clause 66 wherein the cranial nerve is the opticnerve.

72. The method of clause 66 wherein the cranial nerve is the oculomotornerve.

73. The method of clause 66 wherein the cranial nerve is the trochlearnerve.

74. The method of clause 66 wherein the cranial nerve is the trigeminalnerve.

75. The method of clause 66 wherein the cranial nerve is the abducensnerve.

76. The method of clause 66 wherein the cranial nerve is the facialnerve.

77. The method of clause 66 wherein the cranial nerve is theglossopharyngeal nerve.

78. The method of clause 66 wherein the cranial nerve is the vagusnerve.

79. The method of clause 66 wherein the cranial nerve is the accessoryor spinal-accessory nerve.

80. The method of clause 66 wherein the cranial nerve is the hypoglossalnerve.

81. The method of any one of clauses 53 to 63 wherein the neuronalinjury is a neuropathy.

82. The method of clause 81 wherein the neuropathy is achemotherapy-induced neuropathy.

83. The method of clause 81 or clause 82 wherein the neuropathy is anacute neuropathy.

84. The method of clause 83 wherein the acute neuropathy is due toexternal trauma.

85. The method of any one of clauses 53 to 84 wherein the administrationis an injection.

86. The method of clause 85 wherein the injection is selected from thegroup consisting of intraarticular, intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous injections.

87. The method of clause 85 wherein the injection is an intraarticularinjection.

88. The method of clause 85 wherein the injection is an intravenousinjection.

89. The method of clause 85 wherein the injection is an intramuscularinjection.

90. The method of clause 85 wherein the injection is an intradermalinjection.

91. The method of clause 85 wherein the injection is an intraperitonealinjection.

92. The method of clause 85 wherein the injection is a subcutaneousinjection.

93. The method of any one of clauses 53 to 92 wherein the administrationis performed within 48 hours of occurrence of the neuronal injury.

94. The method of clause 93 wherein the administration is performedwithin 24 hours of occurrence of the neuronal injury.

95. The method of clause 93 wherein the administration is performedwithin 12 hours of occurrence of the neuronal injury.

96. The method of clause 93 wherein the administration is performedwithin 8 hours of occurrence of the neuronal injury.

97. The method of clause 93 wherein the administration is performedwithin 6 hours of occurrence of the neuronal injury.

98. The method of clause 93 wherein the administration is performedwithin 4 hours of occurrence of the neuronal injury.

99. The method of clause 93 wherein the administration is performedwithin 2 hours of occurrence of the neuronal injury.

100. The method of clause 93 wherein the administration is performedwithin 1 hour of occurrence of the neuronal injury.

101. The method of clause 93 wherein the administration is performedbetween about 1 hour to about 12 hours of occurrence of the neuronalinjury.

102. The method of clause 93 wherein the administration is performedbetween about 2 hours to about 6 hours of occurrence of the neuronalinjury.

103. The method of clause 93 wherein the administration is performedbetween about 1 hour to about 2 hours of occurrence of the neuronalinjury.

104. The method of any one of clauses 53 to 103 wherein theadministration is performed as a single dose administration.

105. The method of any one of clauses 53 to 103 wherein theadministration is performed as a multiple dose administration.

106. The method of any one of clauses 53 to 105 wherein thetherapeutically effective amount of the hydrophobically modifiednanoparticle is from about 1 pg/kg to about 10 μg/kg.

107. The method of clause 106 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 1pg/kg to about 1 μg/kg.

108. The method of clause 106 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 100pg/kg to about 500 ng/kg.

109. The method of clause 106 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 1pg/kg to about 1 ng/kg.

110. The method of clause 106 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 1pg/kg to about 500 pg/kg.

111. The method of clause 106 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 100pg/kg to about 500 ng/kg.

112. The method of clause 106 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 100pg/kg to about 100 ng/kg.

113. The method of clause 106 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 1ng/kg to about 10 mg/kg.

114. The method of any one of clauses 53 to 105 wherein thetherapeutically effective amount of the hydrophobically modifiednanoparticle is from about 1 ng/kg to 1 mg/kg.

115. The method of clause 106 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 1ng/kg to about 1 μg/kg.

116. The method of clause 106 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 1ng/kg to about 500 ng/kg.

117. The method of clause 106 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 100ng/kg to about 500 μg/kg.

118. The method of any one of clauses 53 to 105 wherein thetherapeutically effective amount of the hydrophobically modifiednanoparticle is from about 100 ng/kg to about 100 μg/kg.

119. The method of any one of clauses 53 to 105 wherein thetherapeutically effective amount of the hydrophobically modifiednanoparticle is from about 1 μg/kg to about 500 μg/kg.

120. The method of any one of clauses 53 to 105 wherein thetherapeutically effective amount of the hydrophobically modifiednanoparticle is from about 1 pg/kg to about 100 μg/kg.

121. The method of any one of clauses 53 to 105 wherein thetherapeutically effective amount of the hydrophobically modifiednanoparticle is from about 1 ng/kg to about 10 mg/kg.

122. The method of clause 121 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 100ng/kg to about 1 mg/kg.

123. The method of clause 122 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 1μg/kg to about 500 μg/kg.

124. The method of clause 122 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 100μg/kg to about 400 μg/kg.

125. The method of any one of clauses 53 to 105 wherein thetherapeutically effective amount of the hydrophobically modifiednanoparticle is about 0.01 μg to about 1000 mg per dose.

126. The method of clause 105 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 1 μgto about 100 mg per dose.

127. The method of clause 126 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 100 μgto about 50 mg per dose.

128. The method of clause 126 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 500 μgto about 10 mg per dose.

129. The method of clause 126 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 1 mgto 10 mg per dose.

130. The method of clause 126 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 1 toabout 100 mg per dose.

131. The method of any one of clauses 53 to 105 wherein thetherapeutically effective amount of the hydrophobically modifiednanoparticle is from about 1 mg to 5000 mg per dose.

132. The method of clause 131 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 1 mgto 3000 mg per dose.

133. The method of clause 131 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 100 mgto 3000 mg per dose.

134. The method of clause 131 wherein the therapeutically effectiveamount of the hydrophobically modified nanoparticle is from about 1000mg to 3000 mg per dose.

135. The method of any one of clauses 53 to 134 wherein thetherapeutically effective amount of the hydrophobically modifiednanoparticle is wherein the method is associated with an improvement ina pharmacokinetic parameter in the patient.

136. The method of any one of clauses 53 to 135 wherein the method isassociated with a reduction in organ toxicity in the patient.

137. The method of any one of clauses 53 to 136 wherein the method isassociated with a reduction in kidney damage in the patient.

138. The method of any one of clauses 53 to 137 wherein the methodreduces a symptom associated with kidney damage.

139. A pharmaceutical formulation comprising the composition of any oneof clauses 1 to 52.

140. The pharmaceutical formulation of clause 139 further comprising apharmaceutically acceptable carrier.

141. The pharmaceutical formulation of clause 139 or clause 140optionally including one or more other therapeutic ingredients.

142. The pharmaceutical formulation of any one of clauses 139 to 141wherein the formulation is a single unit dose.

143. A lyophilisate or powder of the pharmaceutical formulation of anyone of clauses 139 to 142.

144. An aqueous solution produced by dissolving the lyophilisate orpowder of clause 142 in water.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows recovery of locomotor function in SCI rats, measured byBasso Beattie Bresnahan (BBB) score, after intravenous injection of 1 mlof 5 mg/ml curcumin-loaded hydrophobically modified glycol chitosan(HGC) nanoparticles. The loading efficiency is 10%, corresponding to 500μg/ml curcumin in the nanoparticle solution. The injection through thejugular vein was performed at 2 hours after a contusion injury of spinalcord.

FIG. 2 shows pharmacokinetics demonstrating the half-life of HGCnanoparticles in blood.

FIG. 3A-D show an exemplary synthesis of curcumin-loaded HGCnanoparticles. FIG. 3A shows that Fferulic acid is a product of curcuminhydrolysis. FIG. 3B shows a synthetic scheme for conjugation between GCand FA. FIG. 3C is a schematic illustration of curcumin-loaded HGCnanoparticles. FIG. 3D illustrates the results of a solubility test ofcurcumin in PBS without (left) or with HGC (right).

FIG. 4A-C show the photoacoustic membrane poration model. FIG. 4A showsthe photoacoustic membrane poration setup. FIG. 4B shows the results ofa membrane integrity test with calcein AM (green) and propidium iodide(red). After irradiation, the cells in the area within the laser spotwere damaged, labeled with propidium iodide, while the cells out of theirradiation area were still healthy, labeled with calcein. FIG. 4C isa_zoomed-in image showing membrane blebbing of a cell after irradiation.Cell nucleus was labeled by propidium iodide. Bar=10 μm.

FIG. 5 shows a double sucrose gap recording chamber for the recordationof CAPs.

FIG. 6 shows a flowchart of in vivo studies for spinal injury andrepair.

FIG. 7 shows precipitation of loaded curcumin in GC-FA nanoparticleswith degree of substitution (DS)=21 (see right panels). In contrast, theGC-FA with DS=11 was capable of stably encapsulating curcumin (see leftpanels).

FIGS. 8A-E show Glycol chitosan chemically conjugated with ferulic acid(FA), a product of curcumin hydrolysis (FIG. 8A); FIG. 8B shows theaverage diameter of the cucumin-loaded GC-FA nanoparticles bytransmission electron microscopy (TEM); FIG. 8C shows the averagediameter of the cucumin-loaded GC-FA nanoparticles by dynamic lightscattering (DLS); FIG. 8D shows co-localization of fluorescence signalsfrom curcumin (left, green) and Cy5.5-labeled GC-FA (right, red); FIG.8E shows precipitation over one month for the curcumin present in GC-FA.

FIG. 9 shows detection of curcumin and warfarin by their ionizedfragments (m/z=149 for curcumin, m/z=161 for warfarin) in the massspectra.

FIG. 10A shows concentration of curcumin using a calibration curvederived from the ratio between mass intensities of curcumin andwarfarin; FIG. 10B shows the concentration of curcumin in the injuredcord compared to the normal cord; FIG. 10C shows blood retention timedetermined by the one-compartment model; FIG. 10D shows the signalobserved at the lesion site of the spinal cord.

FIG. 11 shows curcumin in GC-FA nanoparticles is mostly eliminatedthrough the kidney.

FIG. 12 shows the half-life of non-modified GC.

FIG. 13 shows the fluorescence intensity at the injured spinal cordcompared to other organs.

FIG. 14A shows the fluorescence signal inside the gray matter that ishighly vulnerable to a contusive injury (see the formation of cavities);FIG. 14B shows the myelin sheath in posterior white matter demonstratesirregular morphology; FIG. 14C shows the myelin sheath near centralcanal demonstrates irregular morphology; FIG. 14D is a highmagnification SRS image of the gray matter that demonstrates clots ofred blood cells; FIG. 14E shows that the myelin sheath in the anteriorwhite matter is highly convoluted.

FIG. 15 shows curcumin enters cells and GC-FA targets the cell membraneafter a 4 hour incubation with GC-FA nanoparticles.

FIG. 16A-D show confocal imaging of the cell membrane attachment ofGC-FA and cellular internalization of curcumin (FIG. 16A); treatmentwith 0.2 mg/ml GC-FA/curcumin significantly reduced the number of PIstained cell (FIG. 16B); GC-FA/curcumin treatment increased the survivalrate from 20% to 95% and GC-FA alone helped rescue the cells by 55% FIG.16C); all three treatments significantly protected PC12 cells in theglutamate damage model (FIG. 16D).

FIG. 17 shows recovery of locomotor function in treated rats.

FIG. 18 shows reduction of levels of magnesium and BUN after GC-FAtreatment.

FIG. 19 shows identification of astrocyte and macrophage/activatedmicroglia via GFAP and ED-1.

FIGS. 20A-F and M-O show: the cavity area indicated by astrocyteboundary in saline treated animals (FIG. 20A); the activated astrocytesand activated microglia the fluorescence of GFAP in the epicenter of thelesion in saline treated animals (FIG. 20B); the activated astrocytesand activated microglia the fluorescence of ED-1 in the epicenter of thelesion in saline treated animals (FIG. 20C); the cavity area indicatedby astrocyte boundary in nanoparticle treated animals (FIG. 20D); theactivated astrocytes and activated microglia the fluorescence of GFAP inthe epicenter of the lesion in nanoparticle treated animals (FIG. 20E);the activated astrocytes and activated microglia the fluorescence ofED-1 in the epicenter of the lesion in nanoparticle treated animals(FIG. 20F); the GFAP fluorescence significantly reduced inGC-FA/curcumin treated group compare to saline treated group(187.38±46.37 vs. 339.37±49.47) (FIG. 20M); the ED-1 fluorescencesignificantly reduced in GC-FA/curcumin treated group compare to salinetreated group (103.20±39.67 vs. 242.35±55.38) (FIG. 20N); the cavityarea significantly decreased in the nanoparticle treated group (1.67±0.5mm²) compared to the saline treated group (5.19±0.92 mm²) (FIG. 20O).

FIG. 21 shows safety analysis of curcumin-loaded GC-FA nanoparticlescompared to saline treatment.

FIG. 22 shows functional recovery demonstrated by GC-FA nanoparticlescompared to saline treatment in the IH impactor model.

FIG. 23 shows dose dependency of GC-FA nanoparticles (1 mg/ml and 2.5mg/ml) compared to saline treatment in the NYU impact model.

FIGS. 24A-B show the neuroprotective effect of GC-FA nanoparticles onprimary spinal cord neurons after glutamate-induced excitotoxicity.(FIG. 24A, left column) Bright field images showed morphological changesof primary spinal cord neurons in treatment conditions of control,glutamate (Glu, 100 μM), Glu+GC (0.1 mg/ml) or Glu+GC-FA (0.1 mg/ml) for24 hours. Yellow and red arrows indicate intact and degenerated axons inthe neurons, respectively. (FIG. 24A, right column) Fluorescence imagesof propidium iodide (PI, red, marker of dead cells) and/or Hoechst(blue, nuclear marker for both survival and dead cells) stained neurons.(FIG. 24B) Quantitative results of percent viability of neurons. Scalebar: 20 μm. *, P<0.05, **, P<0.001

FIG. 25A-D show that treatment with GC-FA nanoparticles improvedhistological outcomes. (FIG. 25A-D) Quantitative comparison of (FIG.25A) intensity of GFAP immunoreactivity, (FIG. 25B) intensity of ED1immunoreactivity, (FIG. 25C) number of SMI31-positive axons, and (FIG.25D) area of luxol fast blue (LFB)-stained myelin at the injuryepicenter at day 28 after SCI and saline or GC-FA treatment.

FIG. 26 shows the reduction of myelin loss using GC-FA nanoparticles.

FIG. 27 shows a reduced cavity achieved with the use of GC-FAnanoparticles.

FIG. 28 shows a reduced cavity achieved with the use of GC-FAnanoparticles, as demonstrated by representative 3D reconstructed imagesand volume quantification of the cavities of saline and GC-FA treatedgroups. *, P<0.05. **, P<0.01; n=3 or 4 per group. Data are expressed asmeans±SEM.

FIG. 29A-C show a graphic representation of GC-FA nanoparticles. FIG.29A provides the chemical structure and schematic illustration of GC-FAnanoparticles. FIG. 29B provides an FT-IR spectrum of GC-FA polymer.FIG. 29C shows the Ssize distribution and TEM image of GC-FAnanoparticles (Scale bar: 300 nm).

FIG. 30 shows confirmation of the conjugation of GC and FA in the GC-FAnanoparticles via NMR analysis.

FIGS. 31A-B show that treatment with GC-FA nanoparticles promotedlocomotor recovery after SCI. FIG. 31A is a schematic diagram ofexperimental design. FIG. 31B shows the BBB locomotor rating scaleperformed in rats that received saline (n=9), methylprednisolone (MP,n=5), and GC-FA (n=10) at 2 hours post SCI. Scores were recorded at day1, 7, 14, and 28 post injury in a blinded manner. Data are expressed asmeans±SEM. *, P<0.05, **, P<0.01.

FIG. 32 shows a new methylprednisolone (MP) delivery strategy usingglycol chitosan (GC), a biocompatible carbohydrate derivative,functioning as a carrier of MP and as an antioxidant through its primaryamine groups.

FIG. 33 shows the structure of GC-MP (GlycolChitosan/Methylprednisolone). Cleavage of the ester bond by bloodesterases releases MP.

FIG. 34 shows the pharmacokinetic curves of MPHS, GC-MP, and GC-MP/MPnanoparticles. Results are shown as the concentration ratio per timepost-injection (hours) of the nanoparticles.

FIG. 35 shows an LC-MS Standard Curve for MP detection. Results areshown as the response ratio (MP/TA) per concentration of MP (ug/ml).

FIGS. 36A-B show rescue of glutamate challenged primary neuron and gliacells as measured by LDH release. Studies with glutamate challengedprimary neuron and glial cells show efficacy of GC-MP/MP and GCMP athigh concentrations and GC-MP/MP at low concentrations (FIG. 36A). MPHSis beneficial at lower concentrations. LDH release from glutamatechallenged (100 umol) primary neuron and glial cells treated with GC,GC-MP, GC-MP/MP (all 1 mg/ml) and MPHS (250 ug/ml) (FIG. 36B).

FIG. 37 shows BBB scoring in a functional recovery pilot study. Resultsare shown as the BBB score measured in animals per day post-injury.Animals treated with GC-MP/MP (5 mg/mL) demontrated greater recoverycompared with saline treatment and MPHS treatment.

DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

As used herein, a “hydrophobically modified nanoparticle” means ananoparticle that has been modified with a hydrophobic moiety. Ananoparticle is understood by those of skill in the art to refer to aparticle having at least one dimension of submicron size.

Various embodiments of the invention are described herein as follows. Inone embodiment described herein, a composition is provided. Thecomposition comprises a hydrophobically modified nanoparticle comprisinga polysaccharide and a pharmacophore, wherein the polysaccharide iscovalently bound to the pharmacophore.

In other embodiments, methods of treatment for a neural injury in apatient are provided. In one illustrative embodiment, the methodcomprises the step of administering to the patient a therapeuticallyeffective amount of the hydrophobically modified nanoparticle. Inanother illustrative embodiment, the method comprises the step ofadministering to the patient a therapeutically effective amount of thehydrophobically modified nanoparticle.

In yet other embodiments, pharmaceutical formulations are provided. Insome illustrative embodiments, the pharmaceutical formulation comprisesthe hydrophobically modified nanoparticle.

In various embodiments described herein, the polysaccharide component ofthe hydrophobically modified nanoparticle described herein can becovalently bound to the pharmacophore. In one embodiment, thepolysaccharide is bound to the pharmacophore via an amide bond. In someembodiments, the covalent bond is an ethylene glycol conjugation.

In some embodiments described herein, the polysaccharide component ofthe hydrophobically modified nanoparticle described herein is chitosan.In other embodiments described herein, the polysaccharide component ofthe hydrophobically modified nanoparticle described herein is a chitosanderivative. As used herein, the term “chitosan derivative” refers to amodification of the natural polysaccharide chitosan. In one embodimentdescribed herein, the polysaccharide component of the hydrophobicallymodified nanoparticle described herein is glycol chitosan. The chitosan,chitosan derivative, and glycol chitosan have molecular weights betweenabout 100 Da and about 1,000,000 Da.

In various embodiments described herein, the pharmacophore component ofthe hydrophobically modified nanoparticle described herein is a fattyacid. As used herein, the term “fatty acid” means a carboxylic acid witha long aliphatic tail, and can be either saturated or unsaturated.Examples of fatty acids are well known in the art, for example thosederived from triglycerides or phospholipids.

In other embodiments described herein, the pharmacophore component ofthe hydrophobically modified nanoparticle described herein is cholanicacid. In yet other embodiments described herein, the pharmacophorecomponent of the hydrophobically modified nanoparticle described hereinis ferulic acid. In other embodiments described herein, thepharmacophore component of the hydrophobically modified nanoparticledescribed herein is a ferulic acid derivative.

In some embodiments described herein, the polysaccharide component ofthe hydrophobically modified nanoparticle is glycol chitosan and thepharmacophore component of the hydrophobically modified nanoparticle isferulic acid. In other embodiments, the nanoparticle has a measureddegree of substitution understood by those of skill in the art to referto the number of ferulic acid per chitosan chain. In some embodiments,the nanoparticle has a degree of substitution of ferulic acid per glycolchitosan (ferulic acid:glycol chitosan chain) selected from the groupconsisting of 5:1, 11:1, and 21:1. In one embodiments, the nanoparticlehas a degree of substitution of ferulic acid per glycol chitosan(ferulic acid:glycol chitosan chain) of 11:1.

In other illustrative embodiments described herein, the compositionfurther comprises a therapeutically effective amount of ananti-inflammatory agent. As used herein, the term “anti-inflammatoryagent” refers to any compound that reduces inflammation in a patientand/or reduces the pain or swelling associated with inflammation.

As used herein, the term “therapeutically effective amount” refers to anamount which gives the desired benefit to an animal and includes bothtreatment and prophylactic administration. The amount will vary from oneanimal to another and will depend upon a number of factors, includingthe overall physical condition of the animal and the underlying cause ofthe condition to be treated.

In some embodiments, the anti-inflammatory agent component of thecomposition is a corticosteroid. In other embodiments, thecorticosteroid is selected from the group consisting of betamethasone,dexamethasone, flumethasone, methylprednisolone, paramethasone,prednisolone, prednisone, triamcinolone, hydrocortisone, and cortisone.In one embodiment, the corticosteroid is methylprednisolone (MP). Inanother embodiment, the corticosteroid is methylprednisolonehemisuccinate (MPHS).

The hydrophobicity of the polysaccharide component of thehydrophobically modified nanoparticle may be specifically modified tooptimize the loading efficiency and intracellular delivery ofanti-inflammatory agent as well the insertion of hydrophobic unimers todamaged membranes. Optimization of the loading efficiency can result inmore efficient delivery of the anti-inflammatory agent to the site ofneed within the body. Furthermore, optimization of the loadingefficiency can result in a targeted delivery of the anti-inflammatoryagent to the site of need within the body and may avoid harmful sideeffects or undesired toxicities to other sites within the body.

In various embodiments described herein, the hydrophobically modifiednanoparticles may have an average diameter in solution of about 10 nm toabout 950 nm, about 100 nm to about 950 nm, about 100 nm to about 500nm, about 100 nm to about 400 nm, about 100 nm to about 200 nm, about200 nm to about 400 nm, about 250 nm to about 350 nm, about 300 nm toabout 400 nm, about 10 nm to about 200 nm, about 10 nm to about 150 nm,about 10 nm to about 100 nm, and about 10 nm to about 50 nm. Thesevarious nanoparticles size ranges are also contemplated where the term“about” is not included. In one embodiment, the hydrophobically modifiednanoparticles may have an average diameter of about 100 nanometers. Inone embodiment, the hydrophobically modified nanoparticles may have anaverage diameter of about 150 nanometers. In one embodiment, thehydrophobically modified nanoparticles may have an average diameter ofabout 200 nanometers. In one embodiment, the hydrophobically modifiednanoparticles may have an average diameter of about 250 nanometers. Inone embodiment, the hydrophobically modified nanoparticles may have anaverage diameter of about 300 nanometers. In one embodiment, thehydrophobically modified nanoparticles may have an average diameter ofabout 320 nanometers. In one embodiment, the hydrophobically modifiednanoparticles may have an average diameter of about 350 nanometers. Inone embodiment, the hydrophobically modified nanoparticles may have anaverage diameter of about 400 nanometers. In one embodiment, thehydrophobically modified nanoparticles may have an average diameter ofabout 450 nanometers. In one embodiment, the hydrophobically modifiednanoparticles may have an average diameter of about 500 nanometers. Inone embodiment, the hydrophobically modified nanoparticles may have anaverage diameter of about 550 nanometers. In one embodiment, thehydrophobically modified nanoparticles may have an average diameter ofabout 600 nanometers. In one embodiment, the hydrophobically modifiednanoparticles may have an average diameter of about 650 nanometers. Inone embodiment, the hydrophobically modified nanoparticles may have anaverage diameter of about 700 nanometers. In one embodiment, thehydrophobically modified nanoparticles may have an average diameter ofabout 750 nanometers. In one embodiment, the hydrophobically modifiednanoparticles may have an average diameter of about 800 nanometers. Inone embodiment, the hydrophobically modified nanoparticles may have anaverage diameter of about 900 nanometers.

In various embodiments described herein, the composition describedherein is a micelle. As used herein, the term “micelle” means anaggregate of amphipathic molecules in water, wherein the nonpolarportions are in the interior and the polar portions are at the exteriorsurface.

In various embodiments, methods of treatment for a neural injury in apatient are provided. In one illustrative embodiment, the methodcomprises the step of administering to the patient a therapeuticallyeffective amount of the hydrophobically modified nanoparticle. Thepreviously described embodiments of the hydrophobically modifiednanoparticle are applicable to the methods described herein.

In some embodiments, the neural injury to be treated by the describedmethods is a spinal cord injury. In other embodiments, the neural injuryto be treated by the described methods is a traumatic brain injury. Inyet other embodiments, the neural injury to be treated by the describedmethods is an acute neuronal injury. In other embodiments, the neuralinjury to be treated by the described methods is a cranial neuronalinjury.

In various embodiments described herein, the neuronal injury to betreated results in hearing loss of the patient. In one embodiment, thehearing loss is due to noise-induced nerve damage. In some embodimentsdescribed herein, the neuronal injury to be treated results in vertigoof the patient. In other embodiments described herein, the neuronalinjury to be treated results in loss of equilibrium of the patient. Inyet other embodiments described herein, the neuronal injury to betreated results in nystagmus of the patient. In some embodimentsdescribed herein, the neuronal injury to be treated results in motionsickness of the patient. In other embodiments described herein, theneuronal injury to be treated results in tinnitus of the patient.

In various embodiments described herein, the neuronal injury is adamaged tympanic membrane. In other embodiments described herein, theneuronal injury is a damaged cranial nerve injury. In some embodiments,the cranial nerve is the vestibulocochlear nerve. In one embodiment, thevestibulocochlear nerve comprises the cochlear nerve. In anotherembodiment, the vestibulocochlear nerve comprises the vestibular nerve.

In yet other embodiments described herein, the cranial nerve is theolfactory nerve. In some embodiments, the cranial nerve is the opticnerve. In other embodiments, the cranial nerve is the oculomotor nerve.In yet other embodiments, the cranial nerve is the trochlear nerve. Insome embodiments, the cranial nerve is the trigeminal nerve. In otherembodiments, the cranial nerve is the abducens nerve. In yet otherembodiments, the cranial nerve is the facial nerve. In some embodiments,the cranial nerve is the glossopharyngeal nerve. In other embodiments,the cranial nerve is the vagus nerve. In yet other embodiments, thecranial nerve is the accessory or spinal-accessory nerve. In someembodiments, the cranial nerve is the hypoglossal nerve.

In various embodiments described herein, the neuronal injury is aneuropathy. In some embodiments, the neuropathy is achemotherapy-induced neuropathy. In other embodiments, the neuropathy isan acute neuropathy. In one embodiment, the acute neuropathy is due toexternal trauma.

In various embodiments, the administration according to the describedmethods is an injection. In some embodiments, the injection is selectedfrom the group consisting of intraarticular, intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous injections. In oneembodiment, the injection is an intraarticular injection. In anotherembodiment, the injection is an intravenous injection. In yet anotherembodiment, the injection is an intramuscular injection. In oneembodiment, the injection is an intradermal injection. In anotherembodiment, the injection is an intraperitoneal injection. In yetanother embodiment, the injection is a subcutaneous injection.

In some embodiments, the administration according to the describedmethods is performed within 48 hours of occurrence of the neural injury.In other embodiments, the administration according to the describedmethods is performed within 24 hours of occurrence of the neural injury.In other embodiments, the administration according to the describedmethods is performed within 12 hours of occurrence of the neural injury.In other embodiments, the administration according to the describedmethods is performed within 8 hours of occurrence of the neural injury.In other embodiments, the administration according to the describedmethods is performed within 6 hours of occurrence of the neural injury.In other embodiments, the administration according to the describedmethods is performed within 4 hours of occurrence of the neural injury.In other embodiments, the administration according to the describedmethods is performed within 2 hours of occurrence of the neural injury.In other embodiments, the administration according to the describedmethods is performed within 1 hour of occurrence of the neural injury.In yet other embodiments, the administration according to the describedmethods is performed between about 1 hour to about 12 hours ofoccurrence of the neural injury. In yet other embodiments, theadministration according to the described methods is performed betweenabout 2 hours to about 6 hours of occurrence of the neural injury. Inyet other embodiments, the administration according to the describedmethods is performed between about 1 hour to about 2 hours of occurrenceof the neural injury.

In other various embodiments, the administration according to thedescribed methods is performed as a single dose administration. In otherembodiments, the administration according to the described methods isperformed as a multiple dose administration.

In various embodiments, the dosages of the nanoparticles can varysignificantly depending on the patient condition and the severity of theneural injury. The effective amount to be administered to a patient isbased on body surface area, patient weight or mass, and physicianassessment of patient condition.

Suitable dosages of the nanoparticles can be determined by standardmethods, for example by establishing dose-response curves in laboratoryanimal models or in humans in clinical trials. Illustratively, suitabledosages of nanoparticles (administered in a single bolus or over time)include from about 1 pg/kg to about 10 μg/kg, from about 1 pg/kg toabout 1 μg/kg, from about 100 pg/kg to about 500 ng/kg, from about 1pg/kg to about 1 ng/kg, from about 1 pg/kg to about 500 pg/kg, fromabout 100 pg/kg to about 500 ng/kg, from about 100 pg/kg to about 100ng/kg, from about 1 ng/kg to about 10 mg/kg, from about 1 ng/kg to 1mg/kg, from about 1 ng/kg to about 1 μg/kg, from about 1 ng/kg to about500 ng/kg, from about 100 ng/kg to about 500 μg/kg, from about 100 ng/kgto about 100 μg/kg, from about 1 μg/kg to about 500 μg/kg, from about 1μg/kg to about 100 μg/kg, from about 1 ng/kg to about 10 mg/kg, fromabout 100 ng/kg to about 1 mg/kg, from about 1 μg/kg to about 500 μg/kg,or from about 100 μg/kg to about 400 μg/kg. In each of theseembodiments, dose/kg refers to the dose per kilogram of a patient's oranimal's mass or body weight.

Also illustratively, suitable dosages of nanoparticles (administered ina single bolus or over time) include about 0.01 μg to about 1000 mg perdose, about 1 μg to about 100 mg per dose, about 100 μg to about 50 mgper dose, about 500 μg to about 10 mg per dose, about 1 mg to 10 mg perdose, about 1 to about 100 mg per dose, about 1 mg to 5000 mg per dose,about 1 mg to 3000 mg per dose, about 100 mg to 3000 mg per dose, orabout 1000 mg to 3000 mg per dose.

In some embodiments described herein, the described methods may beassociated with an improvement in a pharmacokinetic parameter in apatient. In one embodiment, the pharmacokinetic parameter that isimproved is the reduction in organ toxicity in a patient. In anotherembodiment, the pharmacokinetic parameter that is improved is thereduction in kidney toxicity in a patient. In yet another embodiment,the pharmacokinetic parameter that is improved is the reduction inkidney damage in a patient.

In various embodiments, pharmaceutical formulations are provided. In oneillustrative embodiment, the pharmaceutical formulation comprises thehydrophobically modified nanoparticle. The previously describedembodiments of the hydrophobically modified nanoparticle are applicableto the formulations described herein. In some embodiments, thepharmaceutical formulations described herein further comprise apharmaceutically acceptable carrier. In some embodiments, thepharmaceutical formulations described herein further comprise apharmaceutically acceptable diluent. Diluent or carrier ingredients usedin the compositions containing nanoparticles can be selected so thatthey do not diminish the desired effects of the nanoparticle. Examplesof suitable dosage forms include aqueous solutions of the nanoparticles,for example, a solution in isotonic saline, 5% glucose or otherwell-known pharmaceutically acceptable liquid carriers such as alcohols,glycols, esters and amides.

“Carrier” is used herein to describe any ingredient other than theactive component(s) in a formulation. Pharmaceutically acceptablecarriers are determined in part by the particular composition beingadministered, as well as by the particular method used to administer thecomposition (see, e.g., Remington's Pharmaceutical Sciences, 17th ed.1985)). The choice of carrier will to a large extent depend on factorssuch as the particular mode of administration, the effect of the carrieron solubility and stability, and the nature of the dosage form. In oneillustrative aspect, the carrier is a liquid carrier.

As used herein, the term “pharmaceutically acceptable” includes“veterinarily acceptable”, and thus includes both human and animalapplications independently. For example, a “patient” as referred toherein can be a human patient or a veterinary patient, such as adomesticated animal (e.g., a pet).

In some embodiments, the pharmaceutical formulations described hereinoptionally include one or more other therapeutic ingredients. As usedherein, the term “active ingredient” or “therapeutic ingredient” refersto a therapeutically active compound, as well as any prodrugs thereofand pharmaceutically acceptable salts, hydrates, and solvates of thecompound and the prodrugs. Other active ingredients may be combined withthe described nanoparticles and may be either administered separately orin the same pharmaceutical formulation. The amount of other activeingredients to be given may be readily determined by one skilled in theart based upon therapy with described nanoparticles.

In some embodiments, the pharmaceutical formulations described hereinare a single unit dose. As used herein, the term “unit dose” is adiscrete amount of the composition comprising a predetermined amount ofthe described nanoparticles. The amount of the described nanoparticlesis generally equal to the dosage of the described nanoparticles whichwould be administered to an animal or a convenient fraction of such adosage such as, for example, one-half or one-third of such a dosage.

Pharmaceutically acceptable salts, and common methodologies forpreparing pharmaceutically acceptable salts, are known in the art andare included in the definition of the compositions described herein.See, e.g., P. Stahl, et al., HANDBOOK OF PHARMACEUTICAL SALTS:PROPERTIES, SELECTION AND USE, (VCHA/Wiley-VCH, 2002); S. M. Berge, etal., “Pharmaceutical Salts,” Journal of Pharmaceutical Sciences, Vol.66, No. 1, January 1977.

The compositions described herein and their salts may be formulated aspharmaceutical compositions for systemic administration. Suchpharmaceutical compositions and processes for making the same are knownin the art for both humans and non-human mammals. See, e.g., REMINGTON:THE SCIENCE AND PRACTICE OF PHARMACY, (1995) A. Gennaro, et al., eds.,19th ed., Mack Publishing Co. Additional active ingredients may beincluded in the pharmaceutical formulation comprising a nanoparticle, ora salt thereof.

In one illustrative embodiment, pharmaceutical formulations for use witha hydrophobically modified nanoparticle for parenteral administrationcomprise: a) a hydrophobically modified nanoparticle; b) apharmaceutically acceptable pH buffering agent to provide a pH in therange of about pH 4.5 to about pH 9; c) an ionic strength modifyingagent in the concentration range of about 0 to about 300 millimolar; andd) a water soluble viscosity modifying agent in the concentration rangeof about 0.25% to about 10% total formula weight or any combinations ofa), b), c) and d) are provided.

In various illustrative embodiments, the pH buffering agents for use inthe compositions and methods herein described are those agents known tothe skilled artisan and include, for example, acetate, borate,carbonate, citrate, and phosphate buffers, as well as hydrochloric acid,sodium hydroxide, magnesium oxide, monopotassium phosphate, bicarbonate,ammonia, carbonic acid, hydrochloric acid, sodium citrate, citric acid,acetic acid, disodium hydrogen phosphate, borax, boric acid, sodiumhydroxide, diethyl barbituric acid, and proteins, as well as variousbiological buffers, for example, TAPS, Bicine, Tris, Tricine, HEPES,TES, MOPS, PIPES, cacodylate, or MES.

In another illustrative embodiment, the ionic strength modulating agentsinclude those agents known in the art, for example, glycerin, propyleneglycol, mannitol, glucose, dextrose, sorbitol, sodium chloride,potassium chloride, and other electrolytes.

Useful viscosity modulating agents include but are not limited to, ionicand non-ionic water soluble polymers; crosslinked acrylic acid polymerssuch as the “carbomer” family of polymers, e.g., carboxypolyalkylenesthat may be obtained commercially under the Carbopol® trademark;hydrophilic polymers such as polyethylene oxides,polyoxyethylene-polyoxypropylene copolymers, and polyvinylalcohol;cellulosic polymers and cellulosic polymer derivatives such ashydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropylmethylcellulose, hydroxypropyl methylcellulose phthalate, methylcellulose, carboxymethyl cellulose, and etherified cellulose; gums suchas tragacanth and xanthan gum; sodium alginate; gelatin, hyaluronic acidand salts thereof, chitosans, gellans or any combination thereof.Typically, non-acidic viscosity enhancing agents, such as a neutral or abasic agent are employed in order to facilitate achieving the desired pHof the formulation.

In one illustrative aspect, parenteral formulations may be suitablyformulated as a sterile non-aqueous solution or as a dried form to beused in conjunction with a suitable vehicle such as sterile,pyrogen-free water. The preparation of parenteral formulations understerile conditions, for example, by lyophilization, may readily beaccomplished using standard pharmaceutical techniques well known tothose skilled in the art.

The aqueous preparations according to the invention can be used toproduce lyophilisates by conventional lyophilization or powders. Thepreparations according to the invention are obtained again by dissolvingthe lyophilisates in water or other aqueous solutions. The term“lyophilization,” also known as freeze-drying, is a commonly employedtechnique for presenting proteins which serves to remove water from theprotein preparation of interest. Lyophilization is a process by whichthe material to be dried is first frozen and then the ice or frozensolvent is removed by sublimation in a vacuum environment. An excipientmay be included in pre-lyophilized formulations to enhance stabilityduring the freeze-drying process and/or to improve stability of thelyophilized product upon storage. For example, see Pikal, M. Biopharm.3(9)26-30 (1990) and Arakawa et al. Pharm. Res. 8(3):285-291 (1991).

In one embodiment, the solubility of the nanoparticles used in thepreparation of a parenteral formulation may be increased by the use ofappropriate formulation techniques, such as the incorporation ofsolubility-enhancing agents.

In various embodiments, formulations for parenteral administration maybe formulated to be for immediate and/or modified release. Modifiedrelease formulations include delayed, sustained, pulsed, controlled,targeted and programmed release formulations. Thus, a nanoparticle maybe formulated as a solid, semi-solid, or thixotropic liquid foradministration as an implanted depot providing modified release of theactive compound.

The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials. The formulations can also bepresented in syringes, such as prefilled syringes.

While the invention is susceptible to various modifications andalternative forms, specific embodiments will herein be described indetail. It should be understood, however, that there is no intent tolimit the invention to the particular forms described, but on thecontrary, the intention is to cover all modifications, equivalents, andalternatives falling within the scope of the invention.

Example 1 Curcumin Reduces Neuronal Cell Injury and Effectively PromotesFunctional Recovery in SCI Rats

An in vitro study shows that curcumin is effective in reducing cellapoptosis in an H₂O₂-induced PC12 cell injury model. Curcumin-loaded,hydrophobically modified glycol chitosan (HGC) nanoparticles wereadministered to a group of five Long Evans rats at two hours aftertraumatic spinal cord injury (SCI). All the rats showed a significantfunctional recovery, as evidenced by an increase of Basso BeattieBresnahan (BBB) locomotor rating score to an average value of 13.8 atday 14. In the control group treated with saline, the average BBB scorewas 6.4 at day 14 (see FIG. 1).

In a separate experiment, the HGC nanoparticles had a blood half-lifetime of 12 hours (see FIG. 2). The enhanced circulation time of the HGCnanoparticles ensures the delivery of the carrier and drug to the siteof injury. These data show encouraging evidence that an extendedtherapeutic time window is achievable by using self-assemblednanostructure of amphiphilic polymer encapsulated with curcumin.

Example 2 Preparation and Characterization of Polymer Nanostructures

An effective way to extend the therapeutic time window of micelletreatment is to encapsulate an anti-inflammatory agent into thehydrophobic core of the micelle, so that both primary and secondaryinjuries will be targeted. In parallel, a separate study showed thatmPEG-polyester micelles with different hydrophobic chains exhibiteddifferent efficiencies in restoration of compound action potential,indicating a critical role of the amphiphilic property in membranerepair. Thus, the hydrophobic core of the micelle is designed based ontwo factors: the loading efficiency of the anti-inflammatory agent andthe membrane repair efficacy.

Most of anti-inflammatory drugs that have been applied to SCI treatmentare steroids and derivatives such as glucocoticoid, methylprednisolone,sodium succinate, and naloxone. However, high-dose steroids have beenshown to increase the risk of wound infections, pneumonia, sepsis anddeath in SCI patients due to respiratory complications. Non-steroidalanti-inflammatory drugs are usually enzyme specific or immune selectivewhich requires fundamental discovery of selective enzymes and immunepathways.

Curcumin, isolated from turmeric in Curcuma longa as a traditional foodingredient, has unique properties. In pharmacologic studies, turmericexhibits antitumor, anti-inflammatory, and anti-infectious activitieswith low toxicity. Specifically, curcumin has been shown to inhibittumor necrosis factor (TNF), downregulates interleukin (IL)-1, IL-6,IL-8, and chemokines, increase the expression of intracellularglutathione, suppress lipid peroxidation, and play an antioxidant rolethrough its ability to bind iron. Curcumin has been applied in diseasessuch as Alzheimer's disease, Parkinson's diseases, cancer, and others. Amajor challenge facing clinical application of curcumin is its rapidsystemic elimination. Thus, a stable carrier delivering curcumin to thetarget tissue is needed.

mPEG-polyesters of various molecular weights is prepared using thedialysis method and load curcumin into the hydrophobic core of themicelle. The loading efficiency of curcumin and stability ofcurcumin-micelle complex in serum was characterized. In parallel to theuse of block copolymers, glycol chitosan with the side chains modifiedwith ferulic acid (FA) was synthesized. With an extended blood residencehalf-life, glycol chitosan nanoparticles have been widely used ascarriers of anti-cancer drugs. Because FA is a product of curcuminhydrolysis, the modification is expected to not only introduce theamphiphilicity, but also enhance the loading efficiency of curcuminfollowing the law of similar mutual solubility. Compared to mPEG-PDLLA,the amine groups in chitosan help attach the polymer to the negativelycharged cell membrane, which facilitates insertion of the hydrophobicside chain into a lipid membrane as well as cellular uptake of curcumin.

A. Preparation of Self-Assembled mPEG-Polyester Micelles and Loading ofCurcumin

The mPEG-PCL(poly ε-caprolactone), mPEG-PLGA (poly lactic-glycolicacid), and mPEG-PLA(poly lactic acid) were synthesized by ring openingpolymerization (Liggins et al. (2002) Adv Drug Deliv Rev 54:191-202,incorporated herein by reference). The molecular weight of PLA, PCL,PLGA will be 4000, and the PEG will be 2000, the same as the molecularweight of mPEG-PDLLA used in the pilot study.

To test the membrane repair efficiency as a function ofhydrophilic-lipophilic balance values, mPEG (2000)-PDLLA copolymers withdifferent molecular weights of PDLLA (4000, 8000, 16000 Da) will besynthesized by ring opening polymerization of D,L-lactide. Different D,Llactic acid to methoxy PEG feed ratios will be used to preparemPEG-PDLLA copolymers with varying degrees of D,L lactic acidpolymerization. In all cases, micelles will be prepared by membranedialysis. CMC will be measured by monitoring the fluorescence behaviorof pyrene entrapped in the hydrophobic core of the micelle (Schild etal. (1991) Langmuir 7:665-671, incorporated herein by reference). Thediameter of micelles will be determined by dynamic light scattering. Thenumber average molecular weight of the hydrophobic block is measuredusing the proton peaks' intensity in 1H NMR spectra recorded on a VarianUnity Inova 500NB spectrometer (Palo Alto, Calif.) operated at 500 MHz.

Curcumin is loaded into the core of mPEG-PDLLA micelles throughhydrophobic interactions. The mPEG-PDLLA copolymer and curcumindissolved in acetone or dimethyl sulfoxide (DMSO) are placed in a porousdialysis tubing (Spectra/Pro), followed by dialysis against 4 L ofdistilled water for more than 24 h at 25° C. Feed ratio ofpolymer-to-drug is varied to find maximum drug loading content and bestloading efficiency. The resultant solution is frozen in a −80° C.freezer and dried using a freeze-dryer FD-5N (EYELA, Tokyo, Japan).Fresh curcumin-loaded micelles in solution are made at the day ofapplication by dissolving the freeze-dried powder in a PBS solutionusing sonication.

B. Characterization of Curcumin-Loaded Micelles

1. Micelle Size and Stability

The micelle size is an important parameter which correlates withsolubilizing efficiency and activities in the blood stream. The size ofparticles in the dried state is measured by transmission electronmicroscopy (TEM; Philips CM 10, 80 kV) (Lee et al. (2007)Biomacromolecules 8:202-208, incorporated herein by reference). The sizeof empty micelles or curcumin-loaded micelles in aqueous condition ismeasured by dynamic light scattering (DLS, PDLLS/Batch DLS instrumentconnected to PD2000 DLS detector, Precision Detectors). The stability ofthese micelles is determined by the changes of size as a function oftime in both aqueous water and serum. Zeta potential showing the netcharge of polymer micelles is measured by ZetaPALS (BrookhavenInstruments).

2. Drug Loading Amount and Efficacy

To quantify the enhanced solubility of curcumin in micelle carriers,drug loading amount and efficacy is measured. Drug loading amount isdefined as the weight ratio of the loaded drug to the micelles. Drugloading efficiency is the percent ratio of the drug incorporated intothe micelles to the initial amount of the drug used in themicellization. In brief, 1 mg freeze-dried curcumin-loaded micelles isdissolved in 1 mL DMSO so that micelles will be dissociated and curcuminwill be released. The fluorescence of curcumin is measuredspectrophotometrically at 427 nm using a UV spectrometry (Spectra MaxM5, Molecular Devices). The drug loading amount and loading efficacy iscalculated based on a set of standard samples containing predeterminedamounts of curcumin.

3. Drug Release

After intravenous injection, curcumin will be released in blood wherethe lipophilic components (e.g. albumin) act as sink condition. Tomeasure the release kinetics of curcumin, 2 mg/ml curcumin-loadedmicelles are dispersed in a tightened dialysis bag and placed in a glassvial containing 40 mL PBS, pH 7.4, with 15% serum. The glass vial isshaken in a thermostatically water bath maintained at 37° C. during thestudy. Approximately 1.0 mL of release medium is taken at predeterminedtime intervals and the same volume of PBS/Serum is refreshed. Cumulativeamount of released curcumin is measured spectrophotometrically in DMSOand the concentration of the released curcumin is calculated usingstandard curve of curcumin in DMSO. All experiments can be done intriplicate.

Example 3 Preparation and Characterization of HGC Nanoparticles

A. Preparation of Curcumin-Loaded HGC Nanoparticles

To synthesize hydrophobically modified glycol chitosan (HGC)nanoparticles with various molecular weights and hydrophobicities,glycol chitosan (GC) with different molecular weights (250, 100, and 50kDa) will be prepared using an acidic degradation method. Then, the GCwill be hydrophobically modified by conjugation with ferulic acid (FA)that is a product of curcumin hydrolysis (see FIG. 3 a). By controllingthe degree of conjugation of FA to GC, the hydrophobicity will bemodulated. In detail, 50 mg of GC (50, 100, or 250 kDa in molecularweight) is dissolved in 15 ml deionized water, followed by dilution withmethanol (15 ml), and mixing with FA (3.5, 7.0 and 10.5 mg,corresponding to 10, 20, and 30 mol % for the primary amines in GC).Conjugation of the carboxyl group in FA to the amine group in GC isinitiated by adding EDC/NHS that is 1.5 fold molar excess of FA. Theresulting solution is gently vortexed for 24 hours at room temperature,dialyzed (molecular cutoff=12 kDa) for 72 hours against excesswater/methanol (1:4 volume ratio), followed by dialysis againstdeionized water, and the product is lyophilized to obtain HGC (see FIG.3 b).

The solvent evaporation method is used to encapsulate curcumin into theHGC nanoparticle. Both HGC and curcumin are dissolved in a co-solventmade of water and methanol (1:1 volume ratio). With the evaporation ofmethanol, HGC in the aqueous solution is hydrophobically self-assembledinto nanoparticles composed of a hydrophilic shell and a hydrophobiccore (see FIG. 3 c). In detail, HGC (5 mg) is dissolved in deionzedwater (2.5 ml), and mixed with curcumin solution (1.25 mg, 20 wt %) inmethanol (2.5 ml). The methanol in the mixture solution is removed usinga rotary evaporator. Preliminary data showed that FA-conjugated glycolchitosan effectively enhanced the solubility of curcumin in PBS solution(see FIG. 3 d). No precipitation was observed over 1 month for thecurcumin encapsulated in the HGC nanoparticles.

B. Characterization of Curcumin-Loaded HGC Nanoparticles

The molecular weight of acid-degraded GC is measured by gel permeationchromatography (GPC). The degree of conjugation of FA to GC isdetermined by colloidal titration (Kwon et al. (2003) Langmuir19:10188-10193, incorporated herein by reference) and UV absorbance ofFA at 250-350 nm in DMSO. The loading amount and loading efficacy ofcurcumin in HGC will be examined using the same method as previouslydescribed. The measurement of physiochemical properties ofcurcumin-loaded HGC nanoparticles and curcumin release test will beconducted using methods previously described. In addition, x-raydiffraction is used to determine the degree of crystallization ofcurcumin inside the nanoparticle.

Two types of amphiphilic polymers are generated, PEG-polyester andFA-modified glycol chitosans of various molecular weights. A pool ofpolymeric nanostructures exhibiting different hydrophobicity and capableof curcumin loading will be ready for cellular, ex vivo and in vivotesting. In the following sections, “polymeric nanostructures” will beused to refer both PEG-polyester micelles and/or FA-modified HGCnanoparticles. In addition to the DLS measurement, Förster resonanceenergy transfer (FRET) spectroscopy is used to monitor the stability ofmicelles in serum. A FRET pair, DiIC₁₈₍₃₎ and DiOC₁₈₍₃₎, is loaded inmicelles as previously described (Chen et al. (2008) Proc Natl Acad SciUSA 105:6596-6601, incorporated herein by reference). By monitoring theFRET efficiency, release of the core-loaded probes to surrounding mediumis monitored in real time.

Example 4 Determination of Cell Rescue Efficiency of PolymericNanostructures Using a Photoacoustic Membrane Poration Model

The micelles and HGC nanoparticles prepared in Examples 2 and 3 arescreened to identify the nanostructures that are able to rescue theinjured cells over an extended time window and to better understand howhydrophobicity affects the polymer-membrane interactions. Aphotoacoustic membrane poration model is used to mimic the traumaticcell injury. The membrane sealing efficiency is quantified by imagingcellular uptake of fluorescently labeled dextrans of various molecularweights. The cells are assessed by apoptosis and necrosis assays, aswell as inflammation markers.

A. Photoacoustic Membrane Poration Model

A membrane poration model that involves femtosecond (fs) laserirradiation of gold nanorods targeted to the cell surface has beendemonstrated (Tong et al. (2007) Adv Mater 19:3136-3141, incorporatedherein by reference). In this model, the nanorod surface is conjugatedwith a positively charged peptide (octaarginine, R8) for attaching thenanorod to the negatively charged cell surface. Because of the sizeeffect, i.e., the hydrodynamic diameter of these nanorods is c.a. 100nm, the nanorods stay on the cell surface for at least one hour beforeentering the cells. Laser irradiation of the nanorods produces aphotothermal effect via plasmonic absorption and relaxation of theoptical energy into phonon energy inside the nanorods. The thermalexpansion of the nanorods generates a burst acoustic (or mechanic) wavethat compromises the integrity of the cell membrane. The poration ofplasma membrane leads to an influx of Ca²⁺ into cells and a subsequentactivation of calpain which degrades the cytosketon and causes blebbingof plasma membrane. The injured cells can be labeled by propidiumiodide, a necrosis marker (see FIG. 4). This process highly mimics thedamage of neuronal cell membrane after a trauma injury. Without laserirradiation, we have previously shown that R8-conjugated nanorods(R8-NRs) caused no toxicity to cells.

To use this model for screening of membrane repair agents, PC12 cellsare grown, as to mimic neuronal cells, in a collagen coated 96-wellplate and incubated with R8-NRs (O.D. 1, 10 μl) for 1 hour. The bindingof R8-NRs on cell surface is confirmed by two-photon luminescence (TPL)imaging before laser irradiation. After washing with PBS, membraneporation is induced by laser irradiation with a fs Ti:sapphire laser(MaiTai HP, Spectra-Physics) having a pulse width of 130 fs and arepetition rate of 80 MHz. The laser is tuned to the wavelength ofplasmon resonance peak of R8-NRs. The formation of pore on plasmamembrane and the pore size are tested by quantifying the cellular uptakeof dextran-FITC with different molecular weight (eg. 4 KDa, 10 KDa, 70KDa). Dextran-FITC is added prior to irradiation. For each type ofdextran-FITC, the irradiation condition (laser energy, irradiation time)is optimized to induce at least 80% of cells permeable. Cells arevisualized using an Olympus FV1000 confocal microscope in Weldon Schoolof Biomedical Engineering.

B. Testing Methods

1. Cell Viability

Cell death is determined using a standard apoptosis kit (Invitrogen)including Alexa Fluor 680 annexin V to indicate early apoptosis andpropidium iodide to label necrosis. A total of 5 μL of Alexa Fluor 680annexin V and 1 μL of propidium iodide (100 μg/mL) are added to thecells after treatment or without treatment as a control as previouslydescribed (Tong et al. (2009) Nanomedicine 4:265-276, incorporatedherein by reference). Independently, a MTT assay is also be performed toquantify the cell death. After laser irradiation and following thetreatment, 10 μL MTT solution (5 mg/mL in PBS) is added to each well ofthe 96 well plate and incubated at 37° C. for 3 hours. After removingthe medium, 200 μL DMSO is added to each well and the optical density isread at 570 nm using a spectrophotometer (SpectraMAX 190, MolecularDevices Corp., CA). Cell viability is assessed 24 hours postphotoacoustic poration.

2. Cell Membrane Integrity

The sealing of cell plasma membrane is tested by addingdextran-rhodamine prior to irradiation and dextran-cy5.5 at differenttime points post-irradiation. Once the cell membrane is repaired, theuptake of dextran-cy5.5 is stopped. The percentage of cell rescue iscalculated by (Nrho positive—Ncy5.5 positive)/Nrho positive, where N isthe number of cells labeled by rhodamine or cy5.5. Images are taken byconfocal microscope and the number of cells is counted by ImageJsoftware.

3. Intracellular Inflammation

Intracellular reactive oxygen species (ROS) is used as a marker ofinflammation. Twenty four hours post-treatment, carboxy-H2DCFDA(Invitrogen) (a ROS indicator) is added to the cells and incubated for30 minutes. Images are taken by confocal microscope. The intensitybetween treated and control groups is compared to characterize theamount of ROS.

4. Experimental Design

To determine the cell rescue efficiency, PC12 cells are divided intofour groups: group 1 containing cells with no photoacoustic poration,group 2 containing cells treated with curcumin-loaded polymericnanostructures after photoacoustic poration, group 3 containing cellstreated with curcumin-free polymeric nanostructures after photoacousticporation, and group 4 containing cells treated with photoacousticporation alone. To examine the effectiveness of polymeric nanostructuresat different lag times between poration and administration, thenanostructures are added into the cell culture solutions at 15 minutes,1 hour, 2 hours, and 6 hours post-photoacoustic poration in group 2 andgroup 3, respectively. Two-way ANOVA test is used to compare theefficiency of different treatments statistically.

Effective nanostructures are identified and the dose response is furtherexamined to provide a reference of dose regimen for ex vivo and in vivostudies. As shown in a previous study (Shi et al. (2010)), themPEG-PDLLA copolymers are effective as low as 3.3 μM when administratedto the spinal tissue, therefore, polymeric nanostructures with unimerconcentration of 0.33 μM, 3.3 μM, 33 μM, and 330 μM will be applied tothe cells cultured in the 96-well plate after photoacoustic poration.

Membrane sealing is believed herein to depend on the amphiphilicproperty of the polymer. A range of polymeric nanostructures may beidentified that are able to seal the damaged membranes and also suppressthe intracellular inflammation via the loaded curcumin. The optimalnanostructures should have good efficacy of cell rescue with a lag timeof at least 2 hours. Because both charge and size affect the diffusionof molecules in a tissue environment, cellular-level effectivenanostructures with different size and charge properties will be testedin Example 5.

An alternative method for membrane poration is by a laser-enabledanalysis and processing (LEAP) apparatus available in Purdue UniversityBindley Bioscience Center.

Example 5 Determination of Functional and Morphological Response of ExVivo Spinal Cord Treated with the Nanoscale Repair Agents

The cellular study in Example 4 provides a means of fast screening of alarge amount of candidate nanostructures. The tissue-level functionaland morphological responses to these nanostructures are determined inthis example. Spinal tissues are more compact and may not be readilyassessable by polymeric nanostructures compared with cell culturecondition. Functional measurements provide important selection criteriafor further in vivo studies. As the example, isolated spinal cords fromadult guinea pigs are compression injured, treated with the candidatenanostructures loaded with curcumin selected in Example 4, and assessedby electrophysiological measurement and morphological studies.

A. Recording of CAP with a Double Sucrose Gap Recording Chamber

Isolation of spinal cord white matter is performed following theprocedures described in (Wang et al. (2005) Biophys J 89:581-591,incorporated herein by reference).

CAPs are recorded using a double sucrose gap recording chamber (see FIG.5). A 4.0 cm long strip of isolated guinea pig spinal cord white matteris supported in the central compartment and continuously perfused withoxygenated Krebs' solution (˜2.0 ml/min) at 37° C. maintained in a waterbath. The free ends of the spinal cord strip are carried through thesucrose gap channels to side compartments filled with isotonic (120 mM)potassium chloride. The white matter strip is sealed on either side ofthe sucrose gap channels, using fragments of plastic coverslip and asmall amount of silicone grease to attach the coverslip to the walls ofthe channel and seal around the tissue. Isotonic sucrose solution (230mM) is continuously running through the gap channels at a rate of 1.0ml/min. The axons are stimulated and CAPs are recorded at opposite endsof the strip of white matter by silver/silver chloride wire electrodespositioned within the side chambers and the central bath. Stimuli, inthe form of bipolar square pulses of 0.1 ms duration, are adjusted tothe smallest amplitude that could produce a full action potential foreach sample.

B. Compression Injury and Treatment

The compression injury will be inflicted by a constant displacement of5-30 sec compression of the spinal cord using modified forcepspossessing a spacer until the CAP drops to 0 mV (Luo et al. (2002) JNeurochem 83:471-480, incorporated herein by reference). For localapplication of micelles, immediately after injury, the spinal cord whitematter strips are kept in perfusing Krebs' solution at the speed of 2.0ml/min. Then the perfusion is stopped and polymeric nanostructures areadded gently to the Krebs' solution in the central compartment at 15minutes, 1 hour, 2 hours, and 4 hours post compression injury, at adesired concentration determined by Example 4. Following the treatmentfor 10 minutes, the spinal cord strips are thoroughly rinsed with Krebs'solution. All the solutions are enriched with 95% 0₂/5% CO₂ throughoutthe experiment.

C. Multimodal NLO Imaging to Monitor Ca²⁺ Entry into Axons

A multimodal NLO microscope has been developed that combined CARS andTPEF on the same platform (Chen et al. (2009) Opt Express 17:1282-1290,incorporated herein by reference). CARS imaging of myelin sheath is usedto define the intra-axonal space. For monitoring calcium entry intoaxons, the spinal sample is pre-incubated in Ca²⁺-free Krebs' solutionfor 30 min, followed by Ca²⁺-free Krebs' solution with 40 μM OregonGreen 488 BAPTA-2 AM (Sigma) for 2 hours. After that, the control groupof healthy spinal cords is incubated in normal oxygenated Krebs' withCa²⁺ for 1 hour; the control group of injured spinal cords arecompressed and then incubated in normal oxygenated Krebs' with Ca²⁺ for1 hour; the nanostructure treated group is compressed and then incubatedfor 1 hour in oxygenated Krebs' solution supplemented with polymericnanostructures at the concentration identified in Aim 2. TPEF signal ofOregon Green will be transmitted through two 520/40 bandpass filters(Ealing Catalog Inc.) and detected by an external photomultiplier tube(H7422-40, Hamamatsu). FluoView software (Olympus, Tokyo, Japan) will beused to merge TPEF and CARS images, and quantify TPEF intensities insideaxons.

D. Measurement of Anti-Inflammatory Response

The anti-inflammatory role of curcumin is tested by western blotting ofIL-1 and caspase 3 level in homogenized spinal tissue. The role ofcurcumin in reducing oxidative stress is determined by measuring theextent of lipid peroxidation and the content of glutathione inside theinjured tissue.

E. Experimental Design

Spinal cord ventral white matter from adult female guinea pigs (350 to500 g body wt) is used. Spinal cords are divided into three groupstreated by curcumin-loaded mPEG-polyester micelle, curcumin-loaded HGC,and saline, respectively. mPEG-polyester micelles and HGCs are tested.The micelles and HGCs are administrated at 15 m minutes, 1 hour, 2hours, and 4 hours post-SCI. These time points will yield atime-dependence curve for each nanostructure. For CAP measurement, 10spinal cords with the length of 4.5 cm each are used to test eachadministration. Spinal cords of 1-cm segments are used for imagingexperiments and measurement of anti-inflammatory response (n=5 pertest).

The plasma membrane damage may also be determined using three moleculeswith different molecular weights: ethidium bromide (EB, MW 400 Da),horseradish peroxidase (HRP, MW 44 kDa, type VI) and lactatedehydrogenase (LDH, MW 140 kDa). EB and HRP are added to the solutionand the uptake of EB and HRP through the membrane breach of the spinaltissue is monitored. The number of EB positive cells and HRP labeledaxons are quantified. LDH is usually confined inside the cell since itis unable to pass through the intact membrane. Therefore, the leakage ofthis enzyme to the extracellular space is indicative of membranedisruption. To detect LDH release, the solution bathing the spinaltissue is collected at the end of each treatment. The spinal tissue isquickly homogenized and the residual tissue LDH will be assessed by alactate dehydrogenase test kit (Sigma, Mo.).

Example 5 will determine the membrane sealing effect andanti-inflammatory effect of the polymeric nanostructures at the tissuelevel. The optimal nanostructures or nanoparticles should have goodefficacy to facilitate the CAP restoration with a lag time of at least 2hours, and can be used in Example 6.

Example 6 Determination of Anatomical and Functional Recoveries Mediatedby Curcumin-Loaded Copolymer Micelles and HGC Nanoparticles Using aContusion SCI Model

A prior study showed the effectiveness of mPEG-PDLLA micelles inrestoring CAP of injured spinal cord white matter tissues at aconcentration that is 10⁵ orders lower than PEG. Moreover, it has beenshown that intravenously administrated mPEG-PDLLA micelles, were able tosignificantly improve locomotor functions in a Long-Evans rat model ofcompression spinal cord injury (see FIG. 1).

To determine the anatomical and functional recovery after SCI, mediatedby mPEG-polyester nanostructures and/or HGC nanoparticles, polymericnanostructures can be administered via tail vein in aclinically-relevant contusive injury model in adult rats, and theoutcomes can be examined by using a combination of physiological,behavioral, and morphological assessments. The flowchart of the in vivostudy is illustrated in FIG. 6, and the methods are detailed below.

A. In Vivo Spinal Cord Injury Model

Computer controlled impact contusion is widely used by the SCI researchcommunity. Briefly, a moderate contusion injury can be induced byweight-drop of a 10 g rod from a height of 12.5 mm using a MulticenterAnimal Spinal Cord Injury Study (MASCIS) spinal cord impactor. Detailedprocedures are described in (Cao et al. (2005) Experimental Neurology191:S3-S16; and Titsworth et al. (2009) Glia 57:1521-1537, bothincorporated herein by reference).

B. Bioavailability Assay

The polymeric nanostructures or the HGC nanoparticles can be deliveredvia tail vein or jugular vein injection. The curcumin-loadednanostructures or nanoparticles are believed to penetrate through thedamaged blood-spinal cord barrier (BSCB) and accumulate at the site ofinjury with high concentrations. To facilitate penetration, ifnecessary, the nanostructures or nanoparticles may be deliveredintrathecally or by direct injection into the cord parenchyma.

Autofluorescent curcumin and cy5.5 labeled copolymers can be used for abioavailability study. Organs including the spinal cord can be extractedat 24 hours after injection of nanostructures and are examined onCaliper IVIS Lumina II which has a spatial resolution of 50 μm. Thebiodistribution of the carrier and curcumin at cellular level can beobserved using a confocal microscope.

For the bioavailability assay, mass spectrometry may also be used todetermine the concentration of curcumin (MW 368) in each extracted organusing isotope-labeled curcumin as an external standard.

C. Behavioral Testing

Effective restoration of the lost locomotor function can be a primaryaim of this example in experimental SCI. The following tests can beperformed to assess different aspects of SCI outcomes.

D. Locomotor Score

A popular and standardized locomotor rating scale is the BBB locomotorrating scale (Basso et al. (1995) Journal of Neurotrauma 12:1-21) whichwas used in the MASCIS. Using the standard BBB paradigm, animals arefirst be pretrained to locomote in an open field that consists of aplastic pool approximately 90 cm in diameter with 7-10 cm-high walls.Two independent examiners study the locomotor ability of each testsubject for approximately 4 minutes, and then rate the subjectlocomotion using a 21-point scale. Following the SCI and treatment bypolymeric nanostructures, the animals can be subsequently testedbeginning as early as 1 day post-treatment with repeated weekly testingroutinely extending to 8 weeks post-treatment.

E. TreadScan Gait Analysis

The TreadScan system measures the forced locomotion, which meets theneeds for gait analysis of animals. Gait analysis allows highlysensitive, noninvasive detection and evaluation of manypathophysiological conditions occurring in SCI. The TreadScan systemtakes video of an animal, running on a transparent belt treadmill usinga high-speed digital camera. The TreadScan system can reliably analyzethe video, and determine various characteristic parameters including thestance time, the swing time, total stride time, stride length, footcontact area size, body-foot spacing distance, foot spacing distances,running speed, stride frequencies, foot coupling measures, and sciaticfunction index related measures such as foot print placement rotationangle with body and toe spread factors. TreadScan outputs the detailedresults of these parameters into Microsoft Excel files and givesstatistical results to meet research requirements.

F. Neuronal Activity Monitoring by Electrophysiology

Somatosensory evoked potential (SSEP) (Kearse et al. (1993) Journal ofClinical Anesthesia 5:392-398; Hurlbert et al. (1993) J Neurotrauma10:181-200, both incorporated herein by reference) can be used toevaluate the loss and recovery of electrophysiological conductionthrough the SCI. The electrophysiological measurements can be performedprior to laminectomy, immediately after compression, and weekly duringthe recovery period. The SSEP represents multisynapse afferentconduction through ascending long tract sensory columns and can beimmediately eliminated by compression of the spinal cord between thesites of stimulation and recording. The stimulation of the tibial nerveof the hindlimb that produces ascending volleys of nerve impulses may berecorded at the contralateral sensory cortex of the brain. Each completeelectrical record can be comprised of separate trains of 200stimulations (<2 mA square wave, 200 μs duration at 3 Hz), offered by aNeuropak 8 stumulator/recorder (Nihon Kohden Inc., Tokyo, Japan) fromsubdermal needle electrodes placed on the skull evoked by bilateralsimultaneous stimulation of the tibial nerve.

G. Morphological Assessment

Morphological assessment using histology can provides the visualevidence of morphological change and recovery in axons, proteins andglial cell activity, which helps in-depth study of SCI pathogenesis andrepair mechanism. The activities of astrocyte and immune cells can beinvestigated using immunohistochemistry. Details of these assays aredescribed in the pilot study (Shi et al (2010)). Additionally,morphological test of myelin loss and intra-axonal spectrin breakdowncan be performed to independently evaluate the recovery. Theanti-inflammatory effects of curcumin can be examined by Westernblotting of IL-1 and caspase 3 in the injured tissue.

H. Assessment of Toxicity

To examine the safety of the nanostructures or nanoparticles, bloodpressure and electrocardiogram can be measured before and afteradministration and subsequent animal body weight is monitored everyother day. For complete blood counts (CBC), 1 ml of blood can becollected from jugular veins every 4 weeks after administration. At theend of locomotor function recovery study, a full gross necropsyexamination can be performed. The weight of liver, spleen and kidney, aswell as of any unusually sized organs, can be recorded. Tissues will befixed in 10% neutral buffered formalin, processed routinely intoparaffin, and 5-μm sections can be stained with haematoxylin and eosin.Liver, spleen, kidney, heart, lung, pancreas, urinary bladder, brain andspinal cord can be examined by light microscopy by a blinded ratveterinary pathologist. Urine samples can be collected every day in thefirst week post injury and once a week afterwards for analysis of pH,glucose, proteins.

I. Experimental Design

Long-Evans rats can be used to examine the effectiveness ofnanostructures or nanoparticles intravenously injected at various lagtimes after the injury. A total of seven groups of rats can be used tocover three lag times (2, 8, and 24 hours) and one control (salineinjection at 2 hours). These time points can be selected based on thetime course for primary injury. The dosage identified to be effectiveduring tissue-level studies can be used. For monitoring locomotorfunction recovery, BBB scores can be recorded by two independentobservers being blind to the treatment (n=15 per group). Forbioavailability assays, Cy5.5-labeled nanostructures or nanoparticlescan be administrated at three lag times (2, 8, and 24 hours) (n=5 pergroup). Acute and chronic toxicity of the polymeric nanostructures atthe dose used for treatment can be assessed (n=10/group). Forimmuno-analysis, the animals can be sacrificed at 2 weeks after thetreatment (n=5 per group). For Western blot assays of IL-1 and caspase3, the animals can be sacrificed at 7 days after the treatment (n=5 pergroup).

This example can identify nanostructures or nanoparticles thateffectively recover the SCI rats when administrated hours after SCI. Adose responsive curve can be established to determine the optimalconcentration of the nanostructures or nanoparticles. The dose can beused for the subsequent determination of the therapeutic time window,which is important for the pre-clinical testing of therapeuticefficacies.

Example 7 Synthesis and Characterization of GC-FA/Curcumin Nanoparticles

A. Methods

The pharmacokinetics of hydrophobically modified glycol chitosan (HGC)nanoparticles is believed to be dependent on the hydrophobicity of thepolymer. Three different molar ratios of ferulic acid (FA) to glycolchitosan (GC) (i.e., 45, 90, and 180) was tested. In all cases, FA wascoupled to GC in the presence of EDAC and NHS in 10 mMHEPES buffer (pH7.2)/DMSO co-solvent. The resulting solution was stirred for 1 day atroom temperature, dialyzed (molecular cutoff=12 kDa) for 3 days againstexcess water/methanol (1v:4v), followed by dialysis against distillwater, and the product was lyophilized to obtain GC-FA conjugates. Thedegree of substitution, defined as the number of FA per one glycolchitosan chain, was determined by UV absorbance of FA at316 nm in DMSO.GC-FA conjugates with three different degrees of substitution (5, 11,and 21 FAs per GC chain) were obtained.

The curcumin loading was based hydrophobic interactions of curcumin withFA. The curcumin was encapsulated into the GC-FA nanoparticles by asolvent evaporation method. Briefly, both GC-FA conjugates and curcumin(20 wt. %) were dissolved in a co-solvent made of water and methanol(1:1 volume ratio). After the evaporation of methanol under vacuum at55° C., the GC-FA in the aqueous solution was self-assembled intonanoparticles.

The loading contents of curcumin in the nanoparticles were determined byUV absorbance of curcumin at 430 nm in DMSO. A larger curcumin loadingefficiency was demonstrated at a higher degree of FA substitution (seeTable 1).

TABLE 1 Curcumin loading efficacy as a function of FA substitutiondegree Curcumin content Sample (wt. %) GC 4.34 GC-FA (DS = 5) 14.26GC-FA (DS = 11) 15.54 GC-FA (DS = 21) 17.62 DS: degree of substitution,indicated by the number of FA units per GC chain

For the GC-FA nanoparticles with degree of substitution (DS)=21, theloaded curcumin precipitated after 1 day incubation in PBS (see FIG. 7).In contrast, the GC-FA with DS=11 was capable of stably encapsulatingcurcumin.

To label Cy5.5 to GC-FA polymer, 1 wt % hydroxysuccinimide ester ofCy5.5 was dissolved in DMSO and mixed with GC-FA solution. The reactionwas performed at room temperature in the dark for 6 hours. Byproductsand unreacted Cy5.5 molecules were removed over a period of two days bydialysis (molecular weight=12 kDa) against distilled water, and theresulting product was lyophilized. The amount of Cy5.5 in the GC-FA wasconfirmed as 0.7 wt %, as determined by absorbance at 690 nm in DMSO.Curcumin was loaded to GC-FA(−Cy5.5) using the same method describedabove.

For biodistribution testing, the nanoparticles were administered toLong-Evans rats after contusion of the spinal cord. Tissue specimensincluding the injured spinal cords were harvested and homogenized at 1hour post-injection. After adding warfarin (0.5 ppm) to the resultantsolution, curcumin in the tissues was extracted by acetone. To quantifythe concentration of curcumin in tissues, paper spray MS was performed.

Curcumin-loaded GC-FA nanoparticles were formed in PBS buffer (pH 7.4)by sonicated using a probe-type sonifier. Nanoparticle sizes andpolydispersity (μ₂/Γ²) were determined using dynamic light scattering(DLS, 90Plus, Brookhaven Instruments Co., NY) at 633 nm and 25° C. Themorphology of the nanoparticles in distilled water (1 mg/ml) wasobserved using transmission electron microscopy (TEM, CM 200 electronmicroscope, Philips). The surface charge in distilled water wasdetermined using a zeta potential analyzer (ZetaPlus, BrookhavenInstruments Co., NY).

Confocal fluorescence images were obtained FV1000 confocal system(Olympus, Tokyo, Japan) equipped with Argon (488 nm) and HeNe (633 nm)lasers and 60×/1.2 NA water objective. Curcumin and GC-FA(−Cy5.5) imageswere acquired with 488 nm and 633 mm excitations, respectively.

For stability testing, curcumin and the nanoparticles were dispersed inPBS (pH 7.4) and incubated at room temperature. The solutions weremonitored for one month.

B. Results

Glycol chitosan (GC, MW 250 kDa) was chemically conjugated with ferulicacid (FA), a product of curcumin hydrolysis (see FIG. 8( a)), tomaximize the curcumin loading efficiency. An encapsulation efficacy of15.54 wt % curcumin was achieved via optimization of the FA conjugationdegree (see Table 1). By transmission electron microscopy (TEM) anddynamic light scattering (DLS), the average diameter of thecucumin-loaded GC-FA nanoparticles (see FIG. 8( b)) were determined tobe 320 nm (see FIG. 8( c)). The polydispersity value (0.207) indicated anarrow size distribution of the nanoparticles. The zeta potential wasmeasured to be 19.5 mV, indicating a positively charged surface of thenanoparticles. Co-localization of fluorescence signals from curcumin(see FIG. 8( d), left, green) and Cy5.5-labeled GC-FA (see FIG. 8( d),right, red) evidenced the encapsulation of curcumin into thenanoparticles. No precipitation was observed over one month for thecurcumin present in GC-FA (see FIG. 8( e) and FIG. 7).

Example 8 Pharmacokinetics and Bio-Distribution of GC-FA/Curcumin

A. Methods

Cy5.5-labeled GC-FA nanoparticles comprising cucurmin (5 mg/1 ml insaline) or curcumin (0.77 mg/1 ml in saline with 0.1 (v/v) % Tween20)was intravenously injected through the jugular vein of rats at 2 hourspost-contusive injury (n=3). Blood samples (100 μl) were drawn throughthe jugular vein at determined times. The blood (50 μl) was mixed with 5μl K3 EDTA as an anticoagulation agent and warfarin (20 ng/4 μl, 0.5ppm) as an internal standard for mass spectrometry analysis. To extractcurcumin, acetone (150 μl) was added to the solution and vortexed for 10minutes. The resulting solution was centrifuged (rpm 5000, 10 min), andthe supernatant was stored at −20° C. until mass spectrometry analysis.To obtain a calibration curve for quantitative analysis, curcumin in ratblood with different concentrations (0-50 ppm) was prepared, and thencurcumin was extracted by the same method described above.

For biodistribution testing of curcumin, the nanoparticles orcurcumin/Tween20 with the same dose of the pharamcokentics study wasadministrated to Long-Evans rats (n=3) at 2 hours after contusion of thespinal cord. Tissue specimens including the injured spinal cord wereharvested at 1 hour post-injection, and the tissues was homogenizedusing a grander. After adding warfain (20 ng/4 μl, 0.5 ppm) to thetissue solution (50 μl), curcumin was extracted by adding acetone (150μl).

To determine the pharmacokinetics and bio-distribution of curcumin,paper spray mass spectrometry was employed. Paper spray massspectrometry analysis was performed using a TSQ Quantum, LTQ ion trap,and ExactiveOrbitrap mass spectrometer.

The blood samples were collected at determined time points using theanticoagulant warfarin. After adding warfarin (0.5 ppm), curcumin in theblood was extracted by mixing with acetone to dissociate thecurcumin-albumin complex. The resulting solution was loaded on achromatography paper. After dropping 100 of methanol to the blood spot,the components in the blood were sequentially ionized by applying a DCvoltage.

The pharmacokinetics and bio-distribution of GC-FA were determined by afluorescence-based analysis. Half of the blood sample (50 μl) collectedin the pharmacokinetics study of curcumin was used for the detection ofCy5.5 in the blood of rats (n=3). GC(−Cy5.5) (5 mg/1 ml in saline) as acontrol group was intravenously administrated to the rats (n=3) at 2hours post-injury and then the blood was drawn by the same method asdescribed above. At 1 day post-injection of curcumin-loadedGC-FA(−Cy5.5) to rats, the rats were sacrificed via transcardialperfusion with saline and the tissues then were harvested. Thefluorescence intensity of Cy5.5 labeled to GC-FA polymer in blood andtissue samples was measured and visualized by a fluorescencespectrometer (SpectraMax M5, Molecular Devices, CA) with excitation at675 nm and emission at 695 nm and IVIS Lumina (Caliper Life Sicences,Inc., MA) with excitation at 640 nm and emission at 695-770 nm. Thequantitative analysis for the bio-distribution of GC-FA polymer wasperformed using the Living Imaging Software (Caliper Life Sciences,Inc., MA).

B. Results

Following injection of Cy5.5-labeled GC-FA nanoparticles, curcumin andwarfarin were detected by their ionized fragments (m/z=149 for curcumin,m/z=161 for warfarin) in the mass spectra (see FIG. 9). Theconcentration of curcumin was obtained by using a calibration curvederived from the ratio between mass intensities of curcumin and warfarin(see FIG. 10( a), insert). To determine whether our formulation couldextend the blood retention time of curcumin, we compared the plasmaconcentration of curcumin between the GC-FA group and the control groupin which the Tween20 surfactant was used as solubilizer of curcumin.Using the one-compartment model, the half-time of curcumin in the bloodfor the Tween20 group and the GC-FA group were measured to be 6 minutesand 36 minutes, respectively.

Biodistribution of curcumin was also studied by mass spectrometry. Itwas determined that curcumin in GC-FA nanoparticles mostly eliminatedthrough the kidney (see FIG. 11). Importantly, the GC-FA groupdemonstrated 6.6 times higher concentration of curcumin in the injuredcord compared to the normal cord (see FIG. 10( b)). In contrast, nodifference was found between normal and injured cords for the Tween20group (see FIG. 10( b)).

In determination of the blood retention time of GC polymers, the GC-FAexhibited a long blood retention time with a half-life of 20 hoursdetermined by the one-compartment model (see FIG. 10( c)). Incomparison, the non-modified GC showed a half-life of 6 hours (see FIG.12).

For the biodistribution assessment, main organs were harvested at 1 dayafter injection and the amount of Cy5.5 fluorescence was quantified byan IVIS instrument. The fluorescence intensity at the injured spinalcord was significantly higher than other organs (see FIG. 13), exceptfor the kidney. Moreover, the strong signal was observed only at thelesion site of the spinal cord (see FIG. 10( d)). Collectively thesedata demonstrate that the hydrophobic modification of GC with FA allowsfor the prolonged circulation of the polymer and enhanced delivery ofboth polymer and curcumin to the injury site.

The distribution of GC-FA was further determined at single cell levelusing a multimodal nonlinear optical microscope that allows stimulatedRaman scattering (SRS) imaging of membranes (green) and two-photonexcitation fluorescence (TPEF) imaging of Cy5.5-labeled GC-FA (red). Thepolymers were found in both the injured white matter and the injuredgray matter. Importantly, a strong fluorescence signal was found insidethe gray matter that is highly vulnerable to a contusive injury(indicated by the formation of cavities) and GC-FA was highlyaccumulated in the gray matter compared to the white matter at 1 daypost injury (see FIG. 14( a)). High magnification SRS image of the graymatter showed clots of red blood cells, indicating blood vessel damagesinduced by contusive impact (see FIG. 14( b), white arrows). GC-FA waspresent in the ventral portion of the dorsal funiculus, close to thecentral canal (see FIG. 14( d)). The white matter was not seriouslydamaged as compare to gray matter (see FIGS. 14( c) and (e)). In fact,the ventral white matter remained morphologically intact with theabsence of fluorescence of Cy5.5 conjugated GC-FA (see FIG. 14( e)).Together these results suggest the targeting of injured spinal cord bythe GC-FA nanoparticles and demonstrated selective accumulation of GC-FAat the lesion site.

Example 9 In Vitro Model

PC12 cells were used as a simple model for neuronal cells to evaluatethe neuroprotective effect of the nanoparticles (as shown previously inFIG. 15, after a 4 hour incubation with GC-FA nanoparticles, curcuminenters cells and GC-FA targets the cell membrane). In this example, PC12cells were incubated for 4 hours with curcumin-loaded GC(−Cy5.5)-FAnanoparticles. Thereafter, the cell membrane attachment of GC-FA andcellular internalization of curcumin were shown by confocal imaging (seeFIG. 16( a)).

Because oxidative stress and glutamate excitotoxicity are two maindifferent pathologies after spinal cord injury [x], the neuroprotectiveeffects of the nanoparticles were further assessed using hydrogenperoxide (H₂O₂) and glutamate-injured PC12 cells. After incubating thecells with GC-FA/curcumin, GC-FA, or curcumin for 4 hours, cellviability was measured by calcein and propidiumlodide (PI) doublestaining

Treatment with 0.2 mg/ml GC-FA/curcumin significantly reduced the numberof PI stained cell (see FIG. 16( b)). GC-FA/curcumin treatment increasedthe survival rate from 20% to 95%, while GC-FA alone helped rescue thecells by 55% (see FIG. 16( c)). In the glutamate damage model, all threetreatments significantly protected PC12 cells (see FIG. 16( d)).Together, these results suggest that the nanoparticles could effectivelyprotect PC12 cells from H₂O₂ and glutamate injuries.

Example 10 In Vivo Spinal Cord Injury Model and GC-FA/CurcuminAdministration

A. Methods

All protocols for this example were approved by the Purdue Animal Careand Use Committee. Adult Long-Evans rats were anesthetized using 90mg/kg ketamine and 5 mg/kg xylazine. A T10 laminectomy was performed toexpose the underlying thoracic spinal cord segment(s). Spinal cordcontusion injury was produced using a weight-drop device developed atNew York University (Tcuner, 1992) and protocol developed by amulticenter consortium (Basso et al., 1996). The exposed dorsal surfaceof the cord was subjected to weight-drop impact using a 10 g rod (2.5 mmin diameter) dropped from a height of 12.5 mm. After the injury, themuscles and skin were closed in layers, and rats were placed on aheating pad to maintain the body temperature of the rats until theyawake. The analgesic buprenorphine (0.05-0.10 mg/kg) was every 12 hoursthrough subcutaneous injection during anaesthesia recovery and for thefirst 3 days post-surgery for pain management post-operation.

Rats were randomly divided into 4 administration groups for comparison:1 ml GC-FA/curcumin (5 mg/ml in saline; n=10); 1 ml GC-FA alone (4 mg/mlin saline; n=8); 1 ml methylprednisolone sodium succinate (MPSS, 30mg/kg; n=5); or an isovolumetric dose of saline (n=10). Treatments wereadministrated 2 hours post-injury by intravenous jugular vein injection.Manual bladder expression was carried out 3 times daily until reflexbladder emptying was established.

The locomotor recovery was assessed using the Basso Beattie Bresnahan(BBB) locomotor rating score. The test was conducted by twoindependently and made an agreement on the score before the scores werefinalized. The BBB score was recorded at day 1, 7, 14, 21, 28post-surgery.

B. Results

The recovery of locomotor function was evaluated and the results areshown in FIG. 17. Significant differences were found at day 7 and overthe following 3 weeks between GC-FA/curcumin treated and MP treatedrats. At day 28, the GC-FA/curcumin group was significantly better thanthe MP group by 6.3 points. Surprisingly, the GC-FA alone group alsoshowed significantly higher score compared to saline control animals atday 14 and the following 2 weeks.

Blood and urine tests were also evaluated in an attempt to understandthe repair mechanism. As shown in Table 2, levels of magnesium and BUN,two important kidney damage indicators, were significantly reduced afterGC-FA treatment (see FIG. 18).

TABLE 2 Blood Test Results Total Protein BUN Creat- Calcium Magnesiumg/dL Mg/dL inine Mg/dL mEq/L Saline Rat 1 5.6 38 0.5 10.7 3.5 treatedRat 2 5.4 38 0.4 10.7 3.4 Rat 3 6.7 40 0.7 10.7 3.3 GC-FA Rat 4 6.5 190.4 10.7 2.7 treated Rat 5 7.4 19 0.6 11.1 2.8 Rat 6 6.3 19 0.4 10.4 2.8Normal range: 5.6-7.6 8.5-22.7 0.2-0.8 5.3-13 1.5-2.5

In addition, GC-FA treatment also reduced the amount of white bloodcells in urine (see Table 3).

TABLE 3 Urine test results Pro- WBC/ Occult Appearance Color tein HPFBlood Saline Rat 1 Turbid Dark Yellow 3⁺ 0-1 2⁺ treated Rat 2 TurbidDark Yellow 3⁺ 2-3 3⁺ Rat 3 Turbid Light Brown 3⁺ 2-3 3⁺ GC-FA Rat 4Cloudy Yellow 1⁺ 0-1 Negative treated Rat 5 Cloudy Light Yellow TraceNone Negative Rat 6 Turbid Dark Yellow 2⁺ None 3⁺

After saline and GC-FA administrations to rats, blood samples werecollected at day 1 and day 28 for acute and chronic toxicity evaluation,respectively. The results of hematology and serum analyses between theGC-FA group and saline treated group were not significantly different.The levels of creatinine and alanine transaminase for the GC-FA groupwere the same as that of the saline group, indicating no damage to thekidney and the liver. The morphology of vital organs was also assessedusing H&E staining. No morphological difference was observed between thegroups treated with saline and GC-FA at day 28 post treatment.

Example 11 Spinal Cord Tissue Preparation and Histological Analysis ofSpinal Cord Tissue Reactivity

A. Methods

Tissue loss and cellular response were also evaluated between theGC-FA/Curcumin treated group and the saline control group. Four weekspost-injury, rats as described in Example 10 were anesthetized andtranscardially exsanguinated with 150 ml physiological saline followedby fixation with 300 ml of ice-cold 4% paraformaldehyde in 0.01 M PBS(PH 7.4). A 1.5-cm thoracic Spinal cord segment at the lesion centerwere carefully dissected and then post-fixed overnight in 4%paraformaldehyde in 0.01 M PBS (PH 7.4), and transferred to 30% sucrosein 0.01 M PBS (pH 7.4). The cord segments were embedded intissue-embedding medium, and 30-μm sagittal sections were cut on afreezing microtome and mounted onto glass slides.

For immunofluorescence staining, the sections were permeabilized andblocked with 0.3% Triton X-100/10% normal goat serum (NGS) in 0.01 M PBS(pH 7.4) for 30 minutes, and primary antibodies were then applied to thesections overnight at 4° C. Glia fibrillary acidic protein (GFAP,diluted 1:220, Abcam) and ED-1 (diluted 1:50; Millipore, St, Charles,Mo., USA) were used as the primary antibody to identify astrocyte andmacrophage/activated microglia (see FIG. 19). The sections wereincubated the following day for 2 hours at room temperature withsecondary antibodies (Alexa Fluor 488, Invitrogen; Cy3, Invitrogen), andwere then washed, mounted, and examined using an Olympus IX70 confocalmicroscope equipped with a Fluo View program. The cavity volume, GFAP,and fluorescence intensity were measured using Image J.

B. Results

The cavity area indicated by astrocyte boundary is shown in FIG. 20( a)and FIG. 20( d), and the activated astrocytes and activated microgliaare shown by the fluorescence of GFAP and ED-1 in the epicenter of thelesion (see FIG. 20( b) and FIG. 20( e)). FIG. 20( o) shows that thecavity area significantly decreased in GC-FA/curcumin treated group(1.67±0.5 mm²) compared to the saline control group (5.19±0.92 mm²) FIG.20( m) and FIG. 20( n) show that the GFAP and ED-1 fluorescencesignificantly reduced in GC-FA/curcumin treated group compare to salinetreated group (187.38±46.37 v.s. 339.37±49.47 for GFAP, 103.20±39.67v.s. 242.35±55.38 for ED-1).

The animals with spinal cord injury but with saline treatment showed anobvious cavity in the white matter on the dorsal side. In contrast,treatment with GC-FA effectively mitigated the white matter loss.

Example 12 Nonlinear Optical Imaging of Spinal Tissues

The injured spinal cord tissue harvested in the biodistribution study ofGC-FA was cross-sectioned at 200 μm thickness using an oscillatingtissue slicer (Electron Microscopy Sciences, Inc., PA). For the SRLimaging, a Ti:sapphire laser (Chameleon Vision, Coherent) of 140 fspulse duration, 80 MHz repetition rate was tuned at 830 nm to pump anoptical parametric oscillator (OPO, APE compact OPO, Coherent). Based onthe C—H molecular vibration, the OPO provided the Stokes beam at ˜1090nm, and then collinearly combined with the pump beam and sent to a laserscanning microscope (BX51, Olympus). The pump and Stokes beam were thenfocused into the sample using a water immersion objective lens (XLPlan N25×, NA 1.05, Olympus). The forward SRL signal was collected by an oilcondenser (U-AAC, NA 1.4, Olympus) and detected by a photodiode(S3994-01, Hamamatsu). The fluorescence signal was collected backwardwith a photomultiplier tube (H7422P-40, Hamamatsu) after an opticalfilter (715/60, Chroma). Pixel dwell time was 4 μs for each image.

Example 13 Safety Analysis of Curcumin-Loaded GC-FA Nanoparticles inRats

Acute and chronic toxicity of the nanoparticles administrated toLong-Evans rats were evaluated by blood and histological analyses. Theanimals were randomized into a nanoparticle-treated group (n=3) or asaline-treated group (n=3). Each animal received either 1.0 ml salinecontaining 5.0 mg curcumin-loaded GC-FA nanoparticles or 1.0 ml puresaline through jugular vein injection. After the treatment, bloodsamples were collected through the jugular vein at day 1 for acutetoxicity analysis, and at day 28 for chronic toxicity analysis.

The results are shown in FIG. 21. Blood counts did not differsignificantly between the two groups. In particular, the levels ofcreatinine and alanine transaminase (ALT) in the nanoparticle group wereat the same level as those in the saline group, indicating no damage tothe kidneys and the liver.

By examination of tissue morphology, the toxicity of the nanoparticlesto main organs was assessed. Organs were harvested at 28 days post thetreatment. No morphological difference was observed between the twogroups (see FIG. 21). Together, these results suggest no adverse effectsin healthy animals after systemic administration of curcumin-loadedGC-FA nanoparticles.

Example 14 Long Term Safety and Efficacy Analysis of Nanoparticles orNanostructures

A long term safety and efficacy study can be performed using any of thenanoparticle or nanostructure embodiments described herein. For example,the long term study can evaluate the safety and efficacy of thehydrophobically modified nanoparticle, the polymeric nanostructure, orthe polysaccharide nanoparticle over a period of one month, over aperiod of two months, or over a longer period of time.

Furthermore, the hydrophobically modified nanoparticle, the polymericnanostructure, or the polysaccharide nanoparticle can be evaluated withor without addition of an anti-inflammatory agent (e.g., curcumin or acorticosteroid such as methylprednisolone).

The safety and efficacy of the nanoparticle or nanostructure can beevaluated at various timepoints over the duration of the study. Forexample, the safety and efficacy evaluation can take place on a daily,weekly, or monthly basis.

The safety and efficacy evaluations can include any of the parametersevaluated in the previous examples, for example the BBB scale and thetoxicity parameters described herein.

Example 15 Functional Recovery Using Nanoparticles in an IH ImpactorModel

The effectiveness of the described nanoparticles can be evaluated in afunctional recovery study of traumatic SCI using an infinite horizon(IH) model. In this example, GC-FA was used as an exemplarynanoparticle. Animals were assigned to one of two groups: one group wasadministered GC-FA (5 mg/ml; 1.0 ml) and the second group wasadministered saline (1.0 ml). Each group contained 6 animals, and theassigned height of the model was 12.5 mm. Each animal received the testarticle via intravenous injection at 2 hours post-injury.

As shown in FIG. 22, animals in the GC-FA group demonstrated asignificant increase in BBB locomotor score at 35 days post-injury. Thisexample confirms the functional recovery of GC-FA nanoparticles inanimals with traumatic SCI using an IH model.

Example 16 Dose Dependencies of Nanoparticles in the NYU Impact Model

The dose dependency of the described nanoparticles can be evaluated in afunctional recovery study of traumatic SCI using the NYU impact model.In this example, GC-FA was used as an exemplary nanoparticle. Animalswere assigned to one of three groups: one group was administered GC-FA(1 mg/ml), a second group was administered GC-FA (2.5 mg/ml), and athird group was administered saline. As shown in FIG. 23, the BBBlocomotor score was evaluated on a weekly basis for all groups up to 49days post-injury.

Example 17 Neuroprotective Effect of Nanoparticles in Glutamate-inducedExcitotoxicity Model

Since glutamate level increase is the most significant pathologicalfeature of SCI, the neuroprotective effect of the describednanoparticles on primary spinal cord neuronal culture was evaluatedusing a glutamate-induced excitotoxicity model. In this example, GC-FAwas used as the exemplary nanoparticle. Four groups were evaluated: 1)control; 2) glutamate administration; 3) glutamate plus GCadministration; and 4) glutamate plus GC-FA administration.

In the control group, spinal cord neurons showed clear neuronal cellbodies and extended neurites (see FIG. 24A, control, yellow arrows).After exposure to glutamate for 24 hours, neuronal loss and breakdown ofneurites were clearly seen (see FIG. 24A, Glu, red arrow). Pretreatmentby GC partially reduced neuronal loss and suppressed neuritedegeneration (see FIG. 24A, Glu+GC, yellow arrow). Pre-treatment byGC-FA nanoparticles showed greater effect on prevention of neuronal lossand neurite disintegration as compared to the use of GC alone (see FIG.24A, Glu+GC-FA, yellow arrow). Neuron viability percentage (%) wasquantified using Hoechst/propidium iodide (PI) staining (see FIG. 24A,right column).

Administration of glutamate for 24 hours led to increasing neuronal lossand only 48% survived the glutamate insult. However, pre-treatments ofGC polymer or GC-FA nanoparticles significantly increased neuronalsurvival by 81% and 98%, respectively (see FIG. 24B). This exampledemonstrates the neuroprotective effect of both GC and GC-FAnanoparticles. The increased survival of neurons after GC-FAnanoparticle treatment compared to treatment with the GC polymer aloneindicates the added neuroprotective effect of FA conjugation.

Example 18 Anatomical Basis of Functional Recovery of Nanoparticles

To determine the anatomical basis of observed functional recovery,several key parameters associated with tissue damage and repair wereevaluated in rats. The evaluated parameters included densities of axons,astrocytes, macrophages, myelin, and volumes of cavity in mice at day 28post injury. In this example, rats were administered either saline(control) or GC-FA as the exemplary nanoparticle.

Astrocytes, which play a major role in the formation of gliosis afterSCI, were visualized using glial fibrillary acidic protein (GFAP)antibodies. The immunoreactivity of GFAP in the GC-FA group was 50% ofthat in the saline group (see FIG. 25A), indicating that GC-FA treatmentreduced astrogliosis at the lesion site. The macrophages play a majorrole in inflammatory responses including modulating axon degenerationand myelin clearance after SCI. As measured by ED1 immunofluorescence,GC-FA treatment decreased the density of macrophages by 24% compared tothe saline treated group (see FIG. 25B).

To determine whether the reduced immunoreactivity of astrocytes andmacrophages benefit the survival of axons and myelin, the densities ofaxons and myelin were evaluated using SMI31 immunofluorescence and luxolfast blue staining, respectively. Compared to the saline treated group,treatment with GC-FA increased the number of spared axons in theepicenter of the spinal cord by 6.6 times (see FIG. 25C) and enlargedthe luxol fast blue stained area by 2 times (see FIG. 25D). Thereduction in myelin loss associated with GC-FA administration is alsoshown in FIG. 26. These results collectively indicate that treatmentwith GC-FA not only suppressed astrogliosis and inflammation, but alsoprotected axons and myelin.

In accordance with the cellular responses described above,administration of GC-FA nanoparticles also reduced the volume of thelesion cavity compared to saline administration (see FIG. 27). Byhematoxylin and eosin (H&E) staining and employment of the Neurolucidasystem, the spinal cord sections were reconstructed into 3D images andthe cavity volume was determined. The cavity volume of the GC-FA treatedgroup was 2.3 times smaller than that of the saline treated group (seeFIG. 28). The reduced cavitation further supports the neuroprotectiveeffect of GC-FA nanoparticles.

Example 19 Physicochemical Characteristics of GC-FA Nanoparticles

To determine the physicochemical characteristics of GC-FA nanoparticles,varying amounts of FA (feed molar ratio of 45-180 mol FA to 1.0 mol GC)were conjugated to GC (M_(w)=250 KDa) (see FIG. 29A). With threedifferent feed ratios of FA, GC-FA polymers with different degree ofsubstitutions of FA were obtained.

The presence of FA in GC-FA polymer was confirmed by characteristicpeaks at 6-8 ppm in ¹H-NMR spectra, and the amide linkage between GC andFA was confirmed by an increase in the amide peak at 1656 cm⁻¹ in FT-IRspectra (see FIG. 29B). Self-assembled GC-FA nanoparticles weregenerated by sonication in aqueous conditions. The zeta-potentials andaverage diameters of GC-FA nanoparticles were measured using azeta-potential dynamic light scattering analyzer. GC-FA nanoparticlesshowed similar positive zeta-potentials, implying the GC shell composesthe nanoparticle surface.

On the other hand, GC-FA nanoparticles with a degree of substitution of12.8 had smaller diameter (236 nm) compared to other nanoparticles, andtheir spherical morphology was confirmed by transmission electronmicroscopy (see FIG. 29C).

In addition, NMR analysis confirms the conjugation of GC and FA in theGC-FA nanoparticles (see FIG. 30).

Example 20 Locomotor Recovery Analysis of Rats AdministeredNanoparticles

To determine the effectiveness of nanoparticles in functional recovery,we employed the Basso Beattie Bresnahan (BBB) locomotor rating scale toassess locomotor recovery in rats. In this example, GC-FA was used asthe exemplary nanoparticle. Rats received intravenous injections ofsaline (control), methylprednisolone (MP) (control), or GC-FA. Allinjections were carried out 2 hours after contusive SCI, as shownschematically in FIG. 31A. The BBB scores were recorded at days 1, 7,14, 21, and 28 after SCI in a blinded manner for all three groups (FIG.31B).

On day 28, an increase of 4.9 points in the BBB scale was observed inthe GC-FA treated group compared to the saline treated group, and anincrease of 5.7 points was observed in the GC-FA treated group comparedto the MP treated group (GC-FA: 14.9±0.7, saline: 10.0±0.7; MP: 9.2±0.2)(see FIG. 31B). The score of 14.9 in the GC-FA treated group indicatesconsistent weight-supported plantar steps and frequent forelimb-hindlimbcoordination, whereas the BBB scores of 9 to 10 in the MP and salinegroups indicate that the rats were only able to achieve weight supportin stance and there was no coordination between fore- and hindlimbs.

Example 21 Preparation of Exemplary Micelle Preparations

Micelles according to the present disclosure can be prepared. Forinstance, in this example, GC-MP/MP micelles are exemplified. GC-MP wasprepared using a carbodiimide cross-linking reaction between thecarboxyl group of methlylprednisolone hemi-succinate (MPHS) and theprimary amine groups on a glycol chitosan (GC) backbone. Variouspercentages (5%, 10%, and 15%) of free amines on the GC were conjugatedwith MPHS, and the percentage of conjugation was analyzed usingUV-absorption spectroscopy. Thereafter, GC-MP was loaded with freemethlylprednisolone (MP) using a roto-evaporator, and the weightpercentage of loaded MP was determined by UV-absorption spectroscopy.Other properties of the micelles, including size, polydispersity, andzeta-potential were also determined. The GC-MP (10%) formulationdemonstrated a desirable combination of small size and high loadingpotential.

Example 22 Pharmacokinetics of Exemplary Nanoparticles

The pharmacokinetics parameters of the described nanoparticles can beevaluated. In this example, the circulation time, clearance, andhalf-life of exemplary GC-MP nanoparticles is evaluated. Preliminarypharmacokinetics data shows that GC-MP has a long circulation time, witha blood half-life of 8 hours (see FIG. 23). GC-MP was conjugated withCy5.5 and the resultant fluorescence was measured in blood samples thatwere taken at various time points following injection in rats (e.g., 5minutes, 15 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 24 hours, and48 hours post-injection). MP delivered by either MPHS or GC-MP/MP showedvery similar circulation periods, as determined by LC-MS analysis ofblood samples collected at various time points following injection inrats (e.g., 5 minutes, 15 minutes, 30 minutes, 1 hour, 3 hours, and 6hours post-injection). In both cases, MP showed rapid clearance inrates, with a blood half-life of about 30 minutes.

Example 23 Detection of Small Quantites of Exemplary Nanoparticles

The detection of a small amount of MP can also be accomplished using anLC-MS technique. The standard curve generated using LC-MS shows a linearrelationship, thus allowing for quantification at very smallconcentrations of MP (see FIG. 24B). In a pilot study using GC-MP/MP,peaks corresponding to MP were detected in samples extracted fromhomogenized spinal tissue.

Example 24 Bioavailability of Exemplary Nanoparticles in an SCI Model

The bioavailability of nanoparticles can be tested in rats in a SCIsetting. For all treatment groups, the bioavailability may be tested attwo time points: 30 minutes following injection of a test article, and24 hours following injection of a test article. The bioavailabilityanalyses may indicate the quantity of how drug may be available as botha burst dose and as an extended release.

A contusive SCI model may be performed in rats using a 10 gram roddropped from a height of 12.5 mm at the 10th thoracic (T10) spinal cordlevel in adult female Sprague Dawley (SD) rats to produce a severe SCIusing an well-established NYU/MASCIS injury device. Injured rats mayreceive an injection of MPHS (0.75 mg, 1 ml saline), GC-MP (5 mg, 1 mlsaline), or GC-MP/MP (0.75 mg MP, 4.25 mg polymer, 1 ml saline) at 2hours post-injury (n=3 per group). Rats may be sacrificed at either 30minutes or 24 hours post-injury, followed by collection of liver,kidney, spleen, lung, injured spinal cord segments, and uninjuredcervical spinal cord segments. The tissue samples may be weighed,homogenized, and internal standard may be added to equal volumes oftissue. MP and internal standard in the spinal cord may be extracted andanalyzed using liquid chromatography mass spectroscopy (LC-MS).

Example 25 Toxicity and Safety of Exemplary Nanoparticles in a Rat Model

The toxicity of nanoparticles can be tested in rats. In this regard,whole blood tests may be performed at day 1, day 7 and day 28 followingadministration of a test article, and histochemical analysis of organsmay be performed at day 28 post-injection. The evaluated animal groupscan be administered test articles in the following groups: GC-MP/MP (5mg, 1 ml saline), GC-MP (5 mg, 1 ml saline), MPHS (30 mg/kg, 1 mlsaline), and a saline control (1 ml).

To examine the safety and toxicity of the nanoparticles, a well-trainedtechnician can measure blood pressure and an electrocardiogram can beperformed before and after treatments. Animal body weights may bemonitored every other day.

For complete blood counts (CBC), 1 ml of blood may be collected from thejugular veins of the rats. The CBC and serum chemistry paneldeterminations can be performed at the Veterinary Clinical PathologyLaboratory (Purdue University).

At 4 weeks following administration, a full gross necropsy examinationof the rats can be performed. The weight of liver, spleen and kidney, aswell as of any unusually sized organs, may be recorded. Tissues can befixed in 10% neutral buffered formalin, processed routinely intoparaffin, and 5 μm sections can be stained with haematoxylin and eosinin Histopathology Service Laboratory (Purdue University). Liver, spleen,kidney, heart, lung, pancreas, urinary bladder, brain and spinal cord ofthe rats can be examined by light microscopy in a blinded manner by arat veterinary pathologist. Urine samples may be collected every day inthe first week post-injury and once a week afterwards for analysis ofpH, glucose, and proteins.

Example 26 Dual Action of Exemplary Nanoparticles in Rescue of GlutamateChallenged Primary Neuron and Glia Cells

Preliminary cell studies in animals administered GC, GC-MP, GC-MP/MP,and MPHS demonstrate significant rescue of glutamate challenged primaryneuron and glia cells, as measured by LDH release at 16 hourspost-administration (see FIG. 25A-D). Animals administered GC-MP orGC-MP/MP demonstrated effective rescue of glutamate challenged primaryneuron and glia cells at a concentration of 1 mg/ml. Although animalsadministered MPHS (250 ug/ml) and GC (1 mg/ml) demonstrated acontribution to rescue as a result of the administered test article, theresults were not significant. Taken together, these results suggest thatMP and GC demonstrate a dual action for providing protective effects inanimals administered GC-MP and GC-MP/MP treatments.

Example 27 Effectiveness of Exemplary Nanoparticles in Pilot In VivoFunctional Recovery Studies

The GC-MP/MP and GC-MP test articles have also been evaluated in pilotin vivo functional recovery studies. As measured by BBB scoring, animalsadministered GC-MP/MP or GC-MP (each 5 mg, 1 ml saline) at about 2 hourspost-contusion injury demonstrated ignificantly greater functionalrecovery compared to administration of MPHS (7 mg, 1 ml saline) orsaline (1 ml) in long-term survival studies of 28 days (see FIG. 26).These results may be expanded to evaluate GC modified with ahydrophobic, but non-bioactive moiety (i.e., “HGC”) to further clarifythe effectiveness of each component.

Example 28 Effectiveness of Exemplary Nanoparticles in Anti-Inflammationand Anti-Apoptosis Studies

The effectiveness of the nanoparticles can be evaluated to examineanti-inflammation and anti-apoptosis properties. In this example, an SCIrat model may be utilized. In this example, rats may be randomlyassigned into one of six groups: 1) Sham+vehicle (saline); 2) SCI+1 mlsaline; 3) SCI+MPHS (7 mg, 1 ml saline); 4) SCI+HGC (5 mg, 1 ml saline);5) SCI+GC-MP (5 mg, 1 ml saline); 6) SCI+GC-MP/MP (0.75 mg MP & 4.25 mgpolymer, 1 ml saline). In all study groups, the test articles may beinjected intravenously through the jugular vein at 2 hour post-injury inthe SCI model. Parameters may be examined at an acute stage (i.e., 24hours post-injury in the SCI model). The 24 hour post-injury timepointmay be selected due to the peak/approximate peak of myeloperoxidase(MPO) activity, TNF-α6, and apoptosis at this juncture.

Methods: Myeloperoxidase (MPO) activity, a marker for neutrophilinfiltration, may be evaluated. MPO activity has been shown to peak at24 hours post-injury in the SCI model. Briefly, the injured spinal cordsegment (8 mm) can be removed from the rat, homogenized, andcentrifuged. The supernatant may be assayed with a buffer containing0.167 mg/ml o-dianisidine and 0.0005% H2O2 using a Biophotometer(Eppendorf) at absorbance of 460 nm. Expression of TNF-α & IL-1β, twomajor cytokines that are involved in post-traumatic inflammation, canalso be evaluated, as well as a DNA ladder (i.e., an indicator ofapoptosis. TUNEL (R&D Systems) method may be used to detect apoptoticcells in vivo.

Example 29 Long-Term Effects of Exemplary Nanoparticles

The effectiveness of the nanoparticles can be evaluated to examine thelong-term effects on tissue protection (lesion area, volume and sparedwhite matter), electrophysiological (transcranial magnetic motor evokedpotentials, tcMMEP), and motor and sensory (Basso Mouse Scale (BMS),elevated gradient beam walking, footprint analysis, and Hargreaves(thermal hyperalgesia) test) recoveries. In this example, rats may berandomly assigned into one of six groups: 1) Sham+vehicle (saline); 2)SCI+1 ml saline; 3) SCI+MPHS (7 mg, 1 ml saline); 4) SCI+HGC (5 mg, 1 mlsaline); 5) SCI+GC-MP (5 mg, 1 ml saline); 6) SCI+GC-MP/MP (0.75 mg MP &4.25 mg polymer, 1 ml saline). In all study groups, the test articlesmay be injected intravenously through the jugular vein at 2 hourpost-injury in the SCI model. Parameters may be examined at up to 6weeks post-injury in the SCI model (i.e., long term survival).

Methods: Histology: Sections can be stained with cresyl violet (CV) andluxol fast blue (LFB), according to existing protocols. Stereologicalanalyses of lesion volume and sparing of white and gray matter tissues,in sections stained with CV and LFB, may be conducted to compareneuroprotection between treatment groups. Transcranial magnetic-motorevoked potentials (tcMMEPs), an in vivo noninvasive electrophysiologicalmeasure of motor pathway function, relies on the activation ofsubcortical structures. Action potentials descend in the ventral spinalcord and synapse onto motoneuron pools in which output signals can berecorded from both of the gastrocnemius muscles using standard methods.Behavioral assessments can include a battery of analyses (describedbelow) at 1, 3, 7, 14, 21, 28, 35 and 42 days post-SCI.

Basso-Bresnahan-Beattie (BBB) locomotor score and subscore for eachanimal can be obtained following a four minute observation session withtwo raters blinded of experiments at each experimental time point.Footprint Analysis may be used to examine the stepping patters of themice according to existing protocols. Rats with frequently orconsistently plantar stepping may be tested (e.g., BBB score ≧13 forboth hindlimbs).

Elevated gradient beam walk: Rats that show frequent or consistentplantar stepping may be evaluated using a graded series of rough metalbeams (≈24 cm long) of various widths: 0.4, 0.8, 1.2, 1.6, and 2.0 cm.The narrowest beam the rats can traverse with less than 10 errors(hindpaw slips or hindquarter falls) can be recorded in four trials.These methods can be chosen because they have been reliably used toassess hind-limb locomotion function, or to monitor central conductionof long pathways after SCI in both animal models and humans. TheHargreaves test (also called the thermal hyperalgesia test) can be usedto assess the withdrawal threshold to paw thermal stimulation accordingto existing protocols.

Example 30 Effectiveness of Exemplary Nanoparticles at VaryingTherapeutic Windows

The therapeutic window for optimal treatment determined in Examples26-99 may be evaluated. In particular, identification of a therapeuticwindow at the acute stage (i.e., up to 24 hours post-SCI) may bedetermined. Specifically, we may test the selected nanoparticlesadministered to animals at 2 hours, 8 hours, and 24 hours post-SCI. Anyof the parameters examined in Examples 6-9 may be utilized in thepresent example, as well as any of the test articles.

What is claimed is:
 1. A composition comprising a hydrophobicallymodified nanoparticle comprising chitosan covalently bound to apharmacophore.
 2. The composition of claim 1 wherein the chitosan iscovalently bound to the pharmacophore via an amide bond.
 3. Thecomposition of claim 1 wherein the chitosan is glycol chitosan.
 4. Thecomposition of claim 1 wherein the pharmacophore is ferulic acid or aferulic acid derivative.
 5. The composition of claim 1 wherein thechitosan is glycol chitosan and the pharmacophore is ferulic acid. 6.The composition of claim 5 wherein the nanoparticle has a degree ofsubstitution of ferulic acid per glycol chitosan (ferulic acid:glycolchitosan chain) in the range of from about 5:1 to about 21:1.
 7. Thecomposition of claim 6 wherein the nanoparticle has a degree ofsubstitution of ferulic acid per glycol chitosan (ferulic acid:glycolchitosan chain) of about 11:1.
 8. The composition of claim 1 furthercomprising a therapeutically effective amount of an anti-inflammatoryagent.
 9. The composition of claim 8 wherein the anti-inflammatory agentis a corticosteroid.
 10. The composition of claim 9 wherein thecorticosteroid is selected from the group consisting of betamethasone,dexamethasone, flumethasone, methylprednisolone, paramethasone,prednisolone, prednisone, triamcinolone, hydrocortisone, and cortisone.11. The composition of claim 10 wherein the corticosteroid ismethylprednisolone.
 12. The composition of claim 1 wherein the averagediameter of the nanostructure is about 10 nm to about 950 nanometers(nm).
 13. The composition of claim 1 wherein the composition is amicelle.
 14. A method of treating a patient having a neuronal injury,the method comprising the step of administering to the patient atherapeutically effective amount of the composition of claim
 1. 15. Themethod of claim 14, wherein the neuronal injury is a spinal cord injury,a traumatic brain injury, or an acute neuronal injury.
 16. The method ofclaim 15 wherein the neuronal injury is a cranial neuronal injury. 17.The method of claim 15, wherein the neuronal injury causes hearing loss,vertigo, loss of equilibrium, nystagmus, motion sickness, or tinnitus.18. The method of claim 14 wherein the administration is by injection.19. The method of claim 18 wherein the injection is selected from thegroup consisting of intraarticular, intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous injections.
 20. Themethod of claim 14 wherein the administration is performed within 48hours of occurrence of the neuronal injury.
 21. The method of claim 14wherein the therapeutically effective amount of the hydrophobicallymodified nanoparticle is from about 10 μg/kg to about 1000 mg/kg.
 22. Apharmaceutical formulation comprising the composition of claim
 1. 23.The pharmaceutical formulation of claim 22 further comprising apharmaceutically acceptable carrier.
 24. The pharmaceutical formulationof claim 23 further comprising one or more additional therapeuticingredients.
 25. The pharmaceutical formulation of claim 22 wherein theformulation is a single unit dose.
 26. A lyophilisate or powder of thepharmaceutical formulation of claim
 22. 27. An aqueous solution producedby dissolving the lyophilisate or powder of claim 26 in water.